Note: Descriptions are shown in the official language in which they were submitted.
CA 02497497 2005-03-02
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REGUT,RTORS FOR ZMPx.ANTATTON
FIEhD OF THE INVENTTON
The present invention relates to a teclrno~.oc~y
utilizing a regulation factor for communication between a
conceptus and mother on implantation for pregnancy. In
particular, the present invention relates to a technology
based on that IP-10 has important functions for conceptus
implantation and in its process.
$ACKGROUND OF Ti~3E INVENTION
Implantation is harmonized processes between a
conceptus and mother including cellular proliferation,
differentiation, and formation of initial placenta. In
ruminants, conceptus is lost at a high rate xn a.n
implantation period (Roberts R.M. et al., (1990) Oxf. Rev.
Reprod . Biol . , 12 : 14'7-180 ) . This may be caused by fa~.lure
in communi,cat~.on between conceptus at~d mother . Once the
communication has been established, the maternal system can
physiologically and immunologically accept the conceptus.
Thus adhesion and invasion (or implantation) of the
conceptus and subsequent placentation will occur.
Interferon-tau (IFN-s) is a protein secreted by
trophoblast cells of ruminants during pert-implantation
period a.nd has an acti~crity inhibi.ti.ng the degeneration of
corpus luteum. IfN-i has an antiwirus activity and
~.nhibxts cellular proliferation s~.m9.J.ax to other
interf~rons. Furthermore, ~FN'-z may have an
immunoregulating activity.
In ruminants, IFN-z is a~ main protein secrdted by
growing conceptus and is involved in the process of
CA 02497497 2005-03-02
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maternal recognition of pregnancy (Martrtl J. et al_, (1979)
J. Raprod . Fsrtil . , 56 : 63--72; Godkin J. D. et al . , ( 3.982 ) ~'.
Reprod. Fertil., 65:141-150: Imakawa K. et al., (7.987)
Nature, 330 : 377-37 9: Stewart H . ,I . et aJ. . , ( 1987 ) J.
Endocrinol., 115:813-815: Roberts R.M. et al., (1992)
Endocr. Rev., x,3:432-452). In o~rines, conceptus starts to
produce zfN- T on Day 8 or 9 of pregnancy. The production
of TFN-'c is the h~.ghest on Day 16, then, decreases
according to attachment of the conceptus on the uterine
endometrium. The conceptus stops the production o~ IFN-z
by Day 22 Or 23 (GUillomot M. et al., (1990) Bio CeIX.,
68:205--21~,; Roberts R.M. et al., (1992) Proc. Soc. Exp.
$iol Med., 200:7-18; Flint A_P_F. et al., (1994) Mol. Cell.
Endocrinol., 100:93-95). IFN-s obstructs ox partially
obstructs degeneration of coxpus luteum by inhibiting
expression of $n estrogen re~aeptor, prevents an oxytocin
receptor from stimulation by estrogen, and suppresses
intermittent release of PGF2a from the endometrium (flXnt
~i.P.F. et al., (1991) J. Reprod. Fertil. (Suppl.), 93:7.3-
25: Spencer T.E. et al., {1995) Endocrinology, 7.36:4932-
4944: Spencex 'I.L. et aZ-, 0.996) Endocrinology, ~.37:~.~.44-
1147). IFN--r controls differentiation of lymphocytes and
production of cytokines (Differentiation of lymplxocytess:
Newton G.R. et aZ., (7.989) Am. ~T. Reprod. Immunol., 19:99-
107: Pontzer C_H. et al., cancer Res., (1991) 5.:5304-5307;
Skopets B. et al., (1992) Vet. Immunol. xmmunopathol.,
34:87.-96; l~sal-Meliani A. et al., (1993) J. Rsprod.
Immunol., 23:149-65, Production of cytokines: Tuo W. et al.,
(7.999) f. Interferom Gytokine Res., 19:79-87; Emond V. at
al . , ( 2000 ) Biol. . Repxod . , 62 : x.728--7.737 ) . xhese show that
=FNTz locally plays an important role in an
immunormgulating process for implantation in ruminant
ungulates.
Interferon- y--inducible protein 10 kDa (IP-10)
bQlongs to a chemokine family that controls inflammmatory
and immune responso~s in many aspaats mainly through
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chemotactic act~.v~,ty toward xubsets of leukocytes. Studies
for mice and humans identified that Ip-10 belongs to C-X-C
chemokine and is induced in various cells such as
macrophages, f~.bxoblasts, astrvcytes, keratinocytes,
epithelial cells, and endothelial cells (Luster A. et al.,
(1985) Nature, 33.5:672-676; Ohmori Y. et al., (1990)
Biochem. Biophys. Res. Commun., 168:1261-1267: Sauty .~. et
al., (1999} J. Tmmuno~.., 7.62:3549-3558; Albanesi C. et al.,
(2000) f. Immunol., 7.66:13951402; Huang D. et al., (2000)
Immunol. Rev., 177:52-67). IE-10 preferentially acts on NIC
cells and activated T cells (phenotype Thl) through C-X--C
chetttokine receptor 3 (CXCR3) .
SUMMARY OF THE INVENTION
8xoohemical communication between conceptus,
which includes embryos and embryonic membranes, and a
maternal system is important far successful implantation
arid subsequent placentation. In particular, since
canceptus is lost at a high rate in implantation pexXod in
ruminants, controlling factors for gestation have been
investigated. Especially, the need exists fdx
investigat~.orx of a maternal factor responding to a signal
from canceptus in the gestation process.
The inventors have diligently ~.nvestigated a
factor contxol~.~.ng pregnancy establishment. In the studies
for determining molecules associated with ovxrie pregnancy,
the ~.nventors have successfully identified a 10 kDa protein
induced by IFN- y(Z1~-10) from cDNA subtraction analysis of
cDNA prepared from RNA extracted from uterine endometrial
tissues of Day 7.7 pxegnant and Day 15 cyclic ewes. Namely,
ovine IP--10, which belongs to a C-X-C chemokine tami,ly and
lacks glutamine-leucine-arginine (EAR) motif, has bean
identified from a cDNA subtraction study between uterine
endometri~ay tissues from Day 1.7 pregnant and Day 15 cyclic
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CW~~.
Noxthexn blot analysis has revealed that a large
amount of IP-10 mRNA are present in early pxegnant
endometx~.um, the amount is highly larger than that in
cyclic endometxium. RT-PCR dGtaction of CXCR3 (a receptor
for IP-10) mRNA expression has revealed that the expression
in pxegnant endometrium is slightly larger than that in
cyclic endometxi.um.
The inventors have determined changes in TFN-z
and IFN- y mRNAS by Northern blott~.ng and RT-PC~t. BEN-r
and Z$N- v may induce the expression of IP-10. The results
have revet~led that ZfN-s and IfN- y mRNAs axe present in
conceptus and pxegnant endometrium at high guantities.
TFN- y mRNPa has been detected in cyclic endometrium.
Tmmunoh~.stoahamiarsl analysis has revealed that both IP-10
and IFN- y protea.ns axe loGnlized in luminal and glandular
epithelium and subepithelial stroma of the uteri. Both IP-
and IFN- y prot~ins are localized in cyclic ewes, but Z1~-
10 staining of the cyclic endometx~.um xs reduced to the
m~.n~,mum. Zn situ hybridization has revealed that IP-10
mRNA is localized in subepithelial stxoma of pregnant uteri.
On the other hand, IP-10 mRNA is not detected .iri cyclic
ewes. IP-10 mRNA expxessxon xr~ manocytes is stimulated in
accordance with the amounts of zfN-a, ZFN-y , and ZFI~T- r ,
and ZFN-T mostly z~aised the amount of IP-10 mRNA. ,F~nalysxs
of cultured endametxxal cells has revealed that IP-10 mRNA
can be stimu3.ated by a small amount of IEN- t . These
results suggest IFN-s induces expression of zPTlO in early
pregnant uteri. Thus, ~.t has been identified that
interaction between rfN-r and IP-10 is important for
immunological communi.catiori between the conceptus and the
maternal tissue. Similar phenomena have been identified on
caprine Ig-10. The xwcrentorr have recognized that a
techno~,oc~y for controlling implantation may be provided by
using IP--10 because TP-10 is widely distx~.buted in various
animals and their homologies are very high, for example,
between ovine and human, and have completed the present
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invention on the basis of the above-mentioned findings.
xn a more specific aspect, the present invention
provides the following:
(1) A pharmaceutical drug and/or animaX drug
comprising an effective amount of one or more members
selected from the group consisting of an IP-10 protein and
IP-~0 analogud5 or IP-10 derivatives having at least a
deletion, addition, and/or substitution of one or more
amino acid residues in IP-10 and having biological
activities essentially equal or equivalent to those of
intact IP-10,
the phaaeutical drug and/or animal drug being an
agent selected from the group consisting of (a) an agent
fox activating canceptus migration, (b) an agent for
promoting conceptus implantation on the uterine wall, (c)
an agent fox treating sterility, (d) an agent for promoting
pregnancy, (e} an agent far controlling interaction between
conceptus and maternal system, (f) an agent for activating
immunoayte migration, and (g) an agent for controlling
immune ~unGtion in the uterus:
(2) The pharmaceutical drug and/or animal drug
according to the above (1), which is derived from mammal
including human, bovine, buffalo, equine, donkey, ovine,
goat, camel, swine, deer, reindeer, yak, canine, cat, and
ape:
(3) A method for attaining a biological activity
selected from the group consisting of (a) activating
conceptus migration, (b) promoting concCptus implantation
on the uterine wall, (c) treating sterility, (d) promoting
pregnancy, (e) controlling interaction between conceptus
and maternal system, (f) activating immunocyte migration,
and (g) controlling immune function in the uterus,
which compxiaaa treating a sample with a material
selected from the group consisting o~ xP-is and IP-10
analogues or IP-10 derivatives having at least a deletion,
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addition, and/or substitution of one or more amino acid
residues in IP-10 arid having biological activa.txes
essentially equal or equ~.valerit to those of intact IP-1.0;
(4) A reagent comprising at least a material selected
from the group consisting of IP--10 and ZP-~.0 analogues or
IP-10 derivatives havxrig at least a deletion, addition,
and/or substitution of one or more am~.no acid residu~s in
IP-3.0 as~d having biolog~.ca1 activities essentially equal or
equivalent to those of intact rF-10 and being useful far
the method according to the above (3);
(5} An assay fox measuring an IP--10 activity to
determine a biological activXty selected from the group
consisting of (a) activating conceptus migration, (b)
promoting conceptus implantation on the uterine wall, (c)
treating stexi.Zi.ty, (d) promoting pregnancy, (e)
controlling interaction between conceptus and maternal
system, (f) aati'crating immunocyte migration, and (g)
controlling immune function in the uterus;
(6) A reagent useful for the assay acaoxding to the
above (5);
(7) ~1 pharmaceutzcal drug and/or animal drug
comprising a nucleic acid having a nual.eotxde sequence
encoding at least a member selected from the group
consisting of an IP-10 protein and IP-10 analogues ox zP-J.0
derivatives hav~.ng at least a deletion, addition, and/or
substitution of one or more amino acid residues in rh-10
and hav~.ng biological activities essentially equal or
equivalent to those of intact TP-10,
the pharmaceutical drug and/or animal drug being an
agent selected from the group consisting of (a~) an agent
fox a~ati~crx~ting conceptus migration, (b) an agent for
promoting conceptus implantation on the uterine wall, (c)
an agent for treating sterility, (d) an agent for promoting
pregnancy, (e} an agent for controlling interaction between
conceptus and maternal system, (f) an agent for activating
immunocytex migration, and (g) an agent for controlling
immune Function in the uterus;
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(8) A pharmaceutical drug and/or animal drug
comprising a nucleic acid selected from the group
consisting of
(i) a nucleotide sequence comprising at least one open
reading frame portion present in SEQ x~ ND: 1 and,
(ii) a nucleotide sequence capable of hybridizing with at
least one sequence described xn the above (i) under a
stringent condition, and
(iii) a nucleotide sequence encoding a peptide containing
an amino acid sequence at least 80~ homologous to a
polypeptide in FIG. 2 or shown by SEQ =D No: 2, wherein
said peptide has a biological activity substantially equal
to that of Ip-10 (for example, ovine xP-lU), including a
biological activity, or an antigenic equivalent thereof,
selected fxom the group consisting of (a) activating
conceptus migration, (b) promoting conceptus implantation
on the uterine wall, (c) treating sterility, (d) promoting
pregnancy, (e) controlling interaction between conceptus
and maternal system, (f) activating immunoayte migration,
and (g) controlling a.mmune function in the uterus,
the pharmaceutical drug and/or animal drug being an
agent seXected from the group consisting of (a) an agent
for activating conceptus migration, (b) an agont for
promoting conceptus implantation on the uterine wall, (c)
an agent for treating steriXity, (d) an agent for promoting
pregnancy, (e) an agent for controlling interaction between
Goriceptus and maternal system, (f) an agent for activating
immunocyte migration, and (g) an agent fox controlling
immune function in the uterus:
(9) A pharmaceutical drug comprising a compound, or
its salt, for promoting ar inhibiting a biological activity
being, for example, selected from (a) activating conceptus
migration, (b) promoting conceptus implantation on the
uterino wall, (c) treating sterility, (d) promoting
pregnancy, (e) controlling intexaction between concoptus
and mntarnal system, (f) activating immunocyte migration,
and (g) controlling immune Function in the uterus,
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said biological activity being owned by
(A) a mat~rial, or its salt, selected from the group
consisting of an IP-10 protein and IP-10 analogues or IP-10
derivatives having at least a deletion, addition, and/ar
substitution of one or more amino acid residues in IP,10
and having biological activities essentially equal or
equivalent to those of intact IP-10, or
(B) a nucleic acid selected from the group consisting
of
(i) a nucleotide sequence comprising at least an
apen rending frame portion present xn sEQ ID NO: 1 and,
(ii) a nucleotide sequence capable of hybridizing
with at least a sequence as defined in the abov~ (i) under
a stringent condition, and
(iii) a nucleotide sequence encoding a peptide
containing an amino acid Sequence at least 80~ hornologvus
to a polypeptide in Fig. 2 ox sh4wn by SEQ ID NO: 2,
wherein said peptide has a biological activity
substantially equal to that of XP-10 (for example, ovine
TP-10), including a biological activity, or an antigenic
equivalent thereof, selected from (a) activating conceptus
migration, (b) pxdmoting conceptus implantation on the
uterine wall, (c) treating sterility, (d) promoting
pregnancy, (e) controlling inte~aation between conceptus
and maternal system, (f) activating immunacyte migration,
and (g) controlling immune function in the uterus;
(10) A method or kit for screening a compound
promoting ox inhibiting a biological activity owned by a
member selected from the group consisting of TP-10, and IP-
analogues ox ZP-10 derivatives having at least a
deletion, addition, and/or substitution of one or more
amino acid xeaiduea in rP-10 and having a biological
activity essentially equal or equivalent to that of intact
xQ-10, or salts thereof, and TP-Z0 nucleic acids encoding
the same, wherein said bioxogical activity is, for ex~unple,
selected trom (a) activating conceptua migration, (b)
promoting adnocptus implantation on the uterine wall, (c)
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treating sterility, (d) promoting pregnancy, (e)
controlling interaction between conceptus and maternal
system, (f) activating immunocyte migration, and (g)
controlling immune function in the uterus,
the method or the kit using or comprisibg (A) a
compound, or its salt, selected from the group consisting
of IP-10 and the IP-10 analogues or zP-10 derivatives or
(8) a material selected from the group consisting of
nucleic acids encoding the dompound in (A), vectors
containing the nucleic acid, and host cells transformed
with tho nucleic acid ox the vector:
(11) The method or kit acavrding to the above (10),
which is useful for screening a compound promoting the
production of IP~10 to prevent development and/or progress
of sterility:
(12) A compound for controlling the production of
IP-10, which is obtained or identified by screening with
the method or kit according to the above (10) or (11):
(13) A reagent for detecting the presence of a mutated
portion capable of altering the activity or expression of
IP-10 wherein said mutated portion is present in IP-10, a
gene encoding IP-10, ox the corresponding RNA, and
genetically diagnosing a disease assogiated with IP-10;
(14) The diagnostic reagent according to the above
(13), which compxi5es at least a material selected from the
group consisting of restriction enzymes which specifically
recognize a mutated portion xn xP-10 gene, mRNA, or hnRNA,.
arid isoschizorners thereof; and oligonucleotide primers
useful for amplification of a gene including a mutated
portion in IP-10 gene, mRNA, or hnRNA; and
(15) A method for genetic diagnosis of a disease
associated with an IP-10 gene which comprises the steps of;
(a) preparing or obtaining a nucleic acid sample,
(b) subjecting the nucleic acid sample in the step (a) to
gene amplification to give amplified nucleic acid fragments
containing one or more mutations potentially present in the
rP-10 gene, and
CA 02497497 2005-03-02
(c) detexzni.rti.rtg the presence o~ mutation in the nuc~.a~,c
acid fragment in the step (c).
zn another aspect. the present invention provides
the following:
(16) A method fo7: dpterminirig or diagnosing a
biological activity in a specimen which comprises
quant~.ta~ting an IP-10 polynucleotide present in the
specimen and using as an indicator the IP-10 polynucleotide
amount to determine or diagnose a biological. activity level
in the specimen wherein said biological activity is
selected from the group eons~,st~.ng of:
(a) activat~.ng aonaeptus migration, (b) promoting conceptus
implantation on the uterine wall, (c} treating sterility,
(d) promoting pregnancy, (e) controlling i.nteraation
between conceptus and maternal system, (f) activating
immunocyte migration, and (g) controlling immune functa.on
in the uterus;
(17) A method for determining or d~.agnosing a
biological activity in a specimen which comprises
quantitating an IP-10 pxotein present in the specimen and
using ~xa an indicator the IP--10 protein amount to detarmirie
or diagnose a biological acti~ctity level in the specimen
wherein said biological. activity is selected from:
(a) activating concoptus migration, (b) promoting conceptus
implantation on the uterine wall, (c) txeating sterility,
(d) promoting pxegrianay, (e} controlling interaction
between conceptus and maternal system, (f) activating
immunocyte migration, and (g) controlling immune function
in the uterus:
t~.8) A composition for determining or diagnosing a
biological activity in a specimen which comprises at least
a member selected from oligonucleotides or polynucleotides
which hybridize with r8-10 golynucleotide under a stringent
condition, and u~ceful fox determining or diagnosing a
biological activity level in the specimen wherein said
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biol,og~.ca~l activity is selected from:
(a) activating conceptus migration, (b) promoting conceptus
implantation an the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) controlling intexactxvn
between conceptus arid maternal system, (f) activating
immunocyte migration, and (g) controlling immun~ function
in the utexus3
(19) A nucleic acid array for determining ox
diagnosing a biological act~.vxty le,crel in a specimen
wherein said biological activity is selected ~xom:
(a) activating conceptus migration, (b) promoting conceptus
implantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) controlling interaction
between conceptus and maternal system, (f) act~.vating
immunocyte migration, and (g) controlling immune function
in the uterus,
the array being provided with (i) an oligonucleotide
ox polynuclootide which hybridizes with IP-10
polynueleot~.de under ~ stringent condition or (ii) IP-10
polynucleotide;
(20) A primer set for PCR amplifying xP-7.0
polynucleotide in a specimen and determining or diagnosing
a biological activity level ~.n a spec~.itten
whexGin said biological activity is selected from:
(a) activating conceptus m~.gxa~ti.on, (b) promoting conceptus
~.mplarlt~tion on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) contxo~.Xing ~.ntexactiori
between ~onceptus and maternal system, (E) activating
immunocyte rni.gratxon, and (g) controlling immune function
in the uterus;
(2z) A diagnosis k~.t fox detexm~.n~.ng ax diagnosing a
biological activity in a specimen which comprises at least
an ar~txbody capable of recognizing IP-10 and useful for
determining or diagnosing a biological activity level in
tha specimen
whexe~,n sa~.d b~.olog~.Ga~l aot~.v~.ty i~ seleoted from:
(a) activating conceptus migration, (b) promoting conceptus
CA 02497497 2005-03-02
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implantat~.vn vn the uterine wall., (c} treating sterility,
(d) promoting pregnancy, (e) controlling interaction
between cona~eptus and maternal. system, (f) activating
3.mmunocyte migxativn, and (g) controlling immune Function
in the uterus:
(22) A diagnosis kit gor determining or diagnosing a
biological activ~.ty in a specimen which compx~.ses at least
elements consist~.ng of
(i.) an immobilized antibody capable of recognizing zP~lO,
and
(ii) n labeled antibody capabJ.e of recognizing IP-10 but
b~.nd~.ng to an epitope different from those targeted by the
antibody (i), and useful for determining or diagnosing s
b~.o~,ogical activity level
wherein rs~xid biological activity is selected from:
(a) activating conceptus migrat~.ori, (b) promoting conceptus
implantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) controlling interaction
between conceptus and matexn~tl systemr (f) activating
immunocyte m~,gxation, and (g) controlling immune function
in the uterus;
(23) A m~thod for measuring a degree of abnormality or
its risk assoc~.ated with a biological activity selected
from the group consisting of:
(a) act~.vati.ng conceptus migration, (b) promoting conceptus
implantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) controlling interaction
between conceptus and maternal system, (f) activating
immunocyte migration, and (g) controlling immune functzon
in the uterus,
the method comprising at least the steps of:
(i) contacting a biological specimen with a support
immob~.l~.Zeti with an antibody capable of recognizing IP-10:
(ii) washing the support contacted with the bi,oXogxca~.
specimenT
(iii) contacting the support contacted with the biological
specimen with a labeled antibody, wherein the labeled
CA 02497497 2005-03-02
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antibody is capable of recognizing an epitope on IP-10
other than that recognized by the immobilized antibody:
( iv) mea~suritig the label on the suppo7rt or boring free;
(v) using the amount of the label measured in the step (iv)
as an indicator of the amount of IP-10 and comparing it
with a result of a normal biologzaal specimen; and
(vi) using the art~o~tnt of TE-~.0 being significantly
different from the result of the normal biological, specimen
as an; itldicator of the degree of abnormality or its risk
associated with the biological activity:
(24) A method for measuring a degree of abnormality
or it$ risk associated with a biologxcsl activity selected
from the group consisting o~:
(a) activat~:ng conceptus migration, (b) promoting conceptus
implantation on the uterine wall, (c) treating sterility,
(d) promotj.rig pregnancy, (e) controlling interaction
between conceptus and maternal system, (f) activating
immunocyte migration, and (g) controlling zmmutne function
in the uterus,
the method comprising at least the steps af:
(i) preparing RNA from a biological spedimen;
(ii) separating the RNA prepared in the step (x) by
eJ.ectrophvresis:
(iii) hybridizing the RNA separated in the step (ii) with a
lt~belmd nucl~otide probe which hybridizes TP-10
polynual,eot~,de under a stringent condition:
(iv) using the amount of the 7.abel hybridized in the step
(i.~.i) as an indicator of expression of IP-10 polynualeotidG
and comparing ~.t w~.th a result of a normal biological
specimen; and
(v) using the amount of expression of Zp-~.0 polynucleotide
being significantly different from the result of the noxma~.
biological specimen as an ir~d~.catox of the degree of
abnorma~~.~.ty or its risk associated with the biological
activity;
(2S) A method for measuring a degree of abnormality or
its xxsk associated with a biological activity selected
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Exam tho group consisting of:
(a) activating conceptus migration, (b) promoting conceptus
implantation on the uterine wall,, (c) treating sterility,
(d) promoting pregnancy, (e) controlling interaction
between conceptus and maternal systmm, (f) activating
immunacyte migration, and (g) controlling immune function
in the uterus,
the method comprising at J.easct the steps of
(i) preparing RNA from a biological specimen;
(ii) generating a first ol7NA strand by using the RNA
prepared in th~ step (i) as a temp~,ate with a dT primer:
(~.ii) amplifying IP--10 polynucleotide by pCR using the cDNA
generated in the step (xi) as et template with a primer set
for amplifying the IP-10 polynucleotide:
(iv) separating the PC~t product in the step (iii) by
electrophoresis;
(v) hybridizing the PCR product separated in the step (iv)
with a labeled nucleotide probe which hybridizes IP-10
polynucleotide under a stx~.ngent condition:
(vi) us~.ng the amount of the label hybridized in the step
(~cr) as an indicator of expression of zP-10 polynucleotide
and comparing it with a result of a normal, biological
specimens and
(vii) us~.ng the amount of expression of IP-10
polynucleotide being significant7.y different from the
result of the normal biological sp~cimen as an indicator o~
the degree of abnormality or its risk assoc~,ated with the
biological activity;
(26) A method fox measuring a degree of abnormality or
~,ts risk associated with a biological activity selected
Pram the group consist~.ng of:
(a) activating conceptus migration, (b) pramCting conceptus
implantat~.on do the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) controlling ~,ntexactivn
between conceptus and maternal system, (f) activating
immunocyte migration, and (g) controlling immune function
in the uterur~,
CA 02497497 2005-03-02
..
the method comprising at least the steps of:
(i) treating a biological specimen ~or tissue fixation;
(ii) sectioning the fixed tissue prepared in the step (i);
(iii) staining the sectioned tissue immunohistologically
with an antibody capable of xecogni2ing ZP-10:
(iv) compaxing the degree of the immunohistological dyed
biological specimen with that of a normal biological
specimen; and
(v) using tha xamount of zP-10 protein being significantly
different from the xesu~.t of the normal biological specimen
as an i.ndi.catax of the degree of abnormality ox its xxsk
associated with the b~.o~,ogiaal activity:
(27) A method for measuring a degree of xxbnormality
or its risk associated with a b~.ological activity selected
from the group consisting of:
(a) activating canceptus migration, (b) promoting conceptus
xmglantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) contxalling interaction
between concegtus and maternal system, (f) acti.~ting
immunocyte migration, and (g) aontro~.~.xng immune function
in the uterus,
the method comprising at least one step selected from
the group consisting of:
(i) amplifying IP-10 polynucleotide ~.ri the biological
specimen by PCR using a primer set fox ampJ.~.fying the rP-10
polynucleotide;
az'~alyzing a nucleic acid fraction separated from the
biological specimen by using (I) an oligonucleat~.de ox
po~.yx~uclevtide which hybrid~.zes with IP--10 polynucleotide
under a stringent condition or (IT) a nucleic acid axray
prov~.ded with IP-10 polynucleotidm: and
(iii) hybrid~.zxnc~ a nucleic acid fraction separated from
the biological specimen with an oligonucJ.eotxde or
poi.ynucleatide which hybridizes with TE--10 polynucleotide
under a stringent condition: and
a step of measuring the amount of IP-10 polynucleoti.de
in the biol.oga.caX speciman, and then comparing the amount
CA 02497497 2005-03-02
-16-
of the zP-10 polynucleotide in the spec.3men w~.th that in a
normal biological specimens
(28) The method according to any one of the abovQ
(23) to (27) for measuring abnormality or its risk
associated with a biol.ogica~. activity selected from the
group consisting of:
(a) activat~.ng conceptus migration, (b) promoting conceptua
implantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) controlling interaction
between conceptus and maternal system, (f) acti,vatxng
immunocyte migration, and (g) controlling immune function
in the uterus,
which comprises quantitatively measuring IP-~0 protein
ox expression of TP-10 pvlynucleotide:
(29) A reagent used in the method according to any
orie of the above (23) to (27) for measuring abnormality or
its risk associated with s biological activity selected
from the group consisting of:
(a) activating conceptus migration, (b) promoting conceptus
implantation on the uterine wall, (e) treating sterility,
(d) promoting pregnancy, (e) control,X~.ng interaction
between conoeptus .and maternal system, (f) activating
immunocyte migration, and (g) controlling immune function
in the uterus,
the reagent containing (i) an antibody capable of
recognizing IP-10, (ii) an antibody capable of binding an
epitape on YP-10 which is different from that recognized by
the antibody (~.), (xxx) an immobilized antibody (i) or
antibody(ii), or (iv) a labeled ant~.body (i) or antibody
t~.~.):
(30) A method for measuring or diagnos~.ng a degree of
abnormality or its risk associated with a biological
~GtlVlty selected from the group consist~.r~g of:
(a) activating cpnceptus migration, (b) promat~.ng conceptus
implantation on the uterine wall, (c) txeati.ng sterility,
(d) promoting pregnancy, (e) controlling interaction
between conGeptus and maternal system, (f) activating
CA 02497497 2005-03-02
_
immunocyte migration, and (g) controlling immune function
in the uterus,
which comprises measux~.ng the amount of IP-10 protein
ox expression of IP-10 polynucleotide in a sample by using
an antibody capable of recognizing IP-10: and
(31) A reagent for measur~.ng ar diagnosing a degree of
abnormality or its risk associated with a b.ioXog~.ca,l
activity selected from the group consisting of:
(a) activating conceptus rnigratian, (b) promoting conceptus
implantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) aantrolling interaction
between conceptus and maternal system, (f) aGt~.vating
immunocyte ma.gxation, and (g) controlling immune funct~.on
in the uterus,
the reagent containing an antibody capab7.e of
recognizing IP-1~ for measuring the amount of IP-14 protein
or expression of xP-x0 polynucleotide in a sample.
The above objectives and other objectzvcs,
features, advantages, and aspacts of the prESent invention
are xoadxly apparent to those sk~.y7.ed in the art from the
follow3.ng disclosures. Tt should be understood, however,
that the description of the specification inc~.udxng' the
following best modes of ca7:xying out the invention,
examples, etc, is illustrating preferred embodiments of the
present invention and given only for explanation thereof.
xt will becomE a~ppa~rent to the skilled in the art that a
great number of variations and/or altgxa-~ions (or
modificationsy of this xn~~rention may be made based on
knowledge from the disclosure in the fo~.J.ow~,ng parts and
other parts of the specification without departing from the
spirit and scope thereof as disclosed hexexn. A11 of the
patent publications and reference documents c~.ted herein
for illustxat~.ve purposes are hereby incorporated by
reference into the present disclosure.
CA 02497497 2005-03-02
I$ ,
BRIEF DESCRIPTION OF THE DR7IWxNGS
FIG. 1 shows a nuClaotide sequence of ovine TP-1D and
ran amino acid sequence deduced from thereof-
FZG. 2 illustrates the comparison of rP-10 mmino acid
sequences of ovine, goat, human, and mouse. Four cyste~.ne
xosidues acre conserved in these animaln, but a glutaunine-
7.eucxne-arginine (ELR) motif preceding the two cysteine
residues from the N-texmXnal is not present. The homology
of ovine IP--~.0 to human IP-10 is n hxghar than that of
mouse TP-1(?. (sCe Table 1).
FTG. 3 shows J.e~crels of IP-10 mRNA expression in the
ovine utexus during implantation examined by Northexn blot
analysis, an electrophoresis pictuxe(the left) and a graph
of densxtometric analysis (the right) of the Noxthexr~ blot
analysis.
FrG. 4 shows an electrophoresis pictuxe (the left) of
the results of semi.quant~.tative PCR of CXCR3 mRNA and G3PDH
mRNA in the uterus of pregnant ewes, and a graph (the
right) of densitometxxc analysis of the swmiquantitative
PCR products fox CXCR3 mRNA and G3PDH mRNA.
FXG. 5 shows levels of IFN~-s and TFN- y mRNA in the
ovine conceptus and utexu~s during implantation, A (the
left) shows the xeaults o~ Northern blot analysis of IFN-r
mRNA in the conceptuses of pregnant ewes, and B (the right)
shows an electrophoxesa.s pzcture (the top) of the results
of semiquantztati.ve PCR of xFN- v mRNA and G3DPH mRNA in tha
ovine uterus and conceptug and a, graph (the bottom) of
densitometric analysis.
FIG. ~ is pictures of biological tissues ~.n
immunoha.stochemical analysis of ovine uterus and conceptus
tissue sections for IP-10 and IFN--y, in which le means
luminal spa.th~lium, ge means glandular epithelium, st means
subep~.thex~.aX stroma, and tr means trophoblast cell: and
the scale bar equals to 100 ,u m.
FTG. 7 is pictures of biological tissues in situ
hybridization analysis of zQ-7.0 in the ovina uterus, in
CA 02497497 2005-03-02
- 1~ -
which le means luminal epithe~.~,um, ge means glandular
epithelium, st moans subepitheiial stroma, and tr means
trvphoblast cell; and the scale bars equal to 40 ~cm in a, c,
and d, and equal, to 7.0 fir. m in b .
FZG. 8 is pictures, in which A shows exectrophoresis
pictures showing effect of zFNa on zP-ZO mRNA levels, B
shows effects of IFNs on TP-10 mRNA levels in the
endometrial exp3ants from cyclic ewes, electrophoresis
pictures (the left in B) of Northern blot analysis of IP-10
mRNA in the endometrial explants stimulated by
predEtermined amounts of IfNa, and a graph (the right in S)
of densitometric analysis of Northern blot analysis of IP-
mRNA and G3pDH mRNA, and C shows an electrophoresis
picture of Western blot analysis of zf--7.0 in the culture
medium from endometrial explants stimulated by TFNs.
fTC. 9 shows effects of IFDT~-r-stimulated endamatrial
culture medium and recombinant IP-~.0 on migratory activity
of pBMCs, in which A shows the results of Western blot
analysis o~ xecambi,riant caprine IP-10 (IP~-10), B shows the -
resuJ.ts of Northern blot analysis of CXGR3 mRNA i.n EsMCa, C
shows migration of PBMCs of TFN-s-treated ax untreated
endom~trial cultures and TP--10, and D shows migration of
PBMCs of IFN- r -treated or rclP-10-txer~ted endometrya~l
Cultures in the presence of an anti-ZP-1a arrtibady_
FIG. 7.0 shows the results of preparation of a
recombinant caprine IP~20 protein and its antibodies, i.n
which A shows the results of SbS-~AG~ of cell lysates,
before (lane 7.) and after (lane 2) purification using a
nickel-cheXat~,ng column, of ~soherichia coli BL21--SI
expressing recombinant caprine TP-10 trcZP-~.0)j 8 shows the
results of Western blot analysis of purified rcIP-10
protein with anti--His--tag antibody {lane 1) and anti-
caprine TP-10 antibody (lane 2); the left in C shows the
results of Northern bXot ana~,xs~.s of CXCR3 mRNA, using RNA
extracted from KU-Z cells that were transiently transfected
with pcDNl~3_7.-caprine CXCR3 (CXCR3) or with parental
pcDNA3.1 (Mock): the right of C snows the resuXts of
CA 02497497 2005-03-02
-20~-
chemotaxis assay of biological activity of rclP--10 to CXCR3
transfected KU-1 cells expressing (Q) and not expressing
(~) CXCR3: and D shows the results o:~ chemotaxis assay of
KU-1 cells in the presence of rclp-J.O, to which pretreated
with the anti-cuprine IP-10 antibody (anti IP-10), no
further treatment applied (-), or pretreated with control
rabbit IgG (control IgG).
FZG. 11 shows expression of IP-10 in the oarly
pregnant caprine utexus, in which A shows the results of
Western blot ana~.ysxs for pr~ssence of IFN--c in the cultu~Ce
medium derived from caprine conceptuses on Days 14 (D14),
Day 17 (D17), and Day 20 (D~0) of pregnancy; H shows the
results o~ Noxthern blot analysis of endometrial IP-10 mRNA
using RNA from pregnant goat (panel left} and from cyclic
goat (panel right) stimulated with rcIFN-s: the le~t of c
shows the results of Westexn blot analysis of IP-10 in the
uterine tlush~,r~g media from cyclic and pregnant goats; the
right o~ C shows densitornetric analysis of Western blot of
IP-10: and D shows in s~,tu hybridiaa~tion analysis of IP-10
mRNA in the aapririe uterus.
FxG. 12 shows expression and cellu~.ar localization of
CXCR3 a.n the caprine conceptuses, in which A shows~,Ievals
of CXCR3 mRNA express~,on exalriiried by RT-PCR: B shows CXCR3
mRNl~1 expression confirmed by Northern blot at~~$7.ysj.s; and C
shows CXCR3 expression cxamin~d by immunofluorescence
analysis using anti-CXCR3 antibody.
FIG. 13 shows binding o~ rcxP-1C1 to the caprine
trophoblast cells, iri which A shows the rate of
biotinylation of recombinant pxoteins; B shows expression
o~ IP-7.0 receptor CXCR3 mRNA and lymphotactin receptor XCRl
mRNA confirmed by RT-PCR: and C shows visualixEd on Day 17
of pregnant caprine conceptuses by usj.ng biotinylated
prote~.n and horseradish peroxidase labeled streptavidin.
FIG. 14 shows stimulation of rcIP-~,0 on the migration
of CXCR3-expressing conceptus cells, in which the left og A
shows the xesults of Northern blot analysis of RNR
extracted from HTS-1: the right of A shows the xesults a~
CA 02497497 2005-03-02
-21-
chemotaxis assay of HTS,~.; B shows effect of rclp-~,a on
migration activity of HTS-1 transfeoted with CXCR3
expression plasmid) and C shows the results of chemotaxis
assay for primary trophoblast ctlls, on Day 17 of pxegnanoy,
expressing CXCR3.
FIG. Z5 shows effect of rcTP-7.0 on the adhesion of
trophoblast cells to fibxonectin os endometrial ep~,the7.:Lal
ce~.ls, in which A shows adhesion of ~aprine trophoblast
cells (on Day 7.7 of pregnancy] stimulated with rcTP-10: B
shows effect of rclP-10 on adhesion of trophoblast cexXs to
fibronect~.n; C shows effect of rclP-1.0 on adhesion of FITS-1
transfected with aaprine CXCR3 expression vector to
ti,bxoneatin; and D shows effect of rcTP-10 on endometrial
epithelial cells for HTS-7. transfectGd with caprine CXCR3
expressxori vector.
FIG.16 shows expression of integrin subunits i.n the
capx~.ne trophoblast cells activated with rcrP-10, in which
upper shows RT-PCR analysis of zntegrin a 5, a V, j3 1, ~ 3,
and (i5 suburiits mRNAs for HTS~1 cells tranafected with
caprine CXCR3 expression vector [2P~10+Abs: rcZQ-7.0 (~0
ng/ml) and pretreatment with anti-IP-1.0 antibody (30 ~ g/ml),
IP-10: rcIP-10 (20 ng/m7.)]; and lower shows densimetric
analysis of PCR products.
BEST MODES OF CARRYING OUT THE xN'VENTZON
In the specification, "zP-~.0" means a 10 kDa
prote~.n 7.nduced by interferon- y ( interferon- y inducible
protein 10 kDa). This protein factor may ind~.cstte x8-J.0
belongs to a chemokine family that controls inflammatory
and immune responses xn many aspects mainly thraugh
chemotactic activity toward subsets of leukocytes. The IP-
may be the protein identified during the studies for
mice and humans as belonging to C-X-C chemvkine and b~ing
induced in a various cells such as macrophages, fibrobl.asts,
astrocytes, kEratinocytes, epithelial cells, and
endothelial celXs. ~'he IP-10 may preferentially act on NK
CA 02497497 2005-03-02
-22-
cells and activated T cel.l.s (phenotype Thl) through C-X-~C
chemokine receptor 3 (CXCR3).
Bovine ZP-7.0 has been particularly investigated
~.n order to identify the specific activities dur~.ng
~.1'np7.antation. Therefore, any ungulate IP-~.0 is included ~,n
the present invention from the viewpoint of these specific
characteristics. furthermore, human IP-1.0, its derivat~.ves,
analogues, equivalents, and the like are included from the
viewpoint of using the act~.v~.txes during implantation.
Ovine IP-10 is a peptide composed of ~,OZ amxrio
acid residues and has four aysteine residues. The four
cysteine residues are conserved within a chemokine family,
and tho first two cysteines are separated by a sa.ngle amj.no
acid residue to form a C-X-C mot~.f. Similar to ~P-lOs of
other animals, ovine IP~10 lacks ELR motif preced~.ng the
first two cysteine residues and has a high similarity to
human zP-10 with 62.7$ homology in the nucleotide sequence
and 75.5 homology in the amino acid sequence. ZP,J.Os
including ovine ZP-10 according to the present invention
have a biological activity selected from the group
consisting of (a) activating conceptus migration, (b)
promoting conceptus implantation on the uterine wall, (c)
txeat~,ng sterility, (d) promoting pregnancy, (e)
controlling interaction between conceptus and maternal
system, (f) activating immunocyte migration, and (g)
controlling immune function in the uterus.
Typically, rP-lOs including ovine IP-10 and
capr~.ne IP,7.0 according to tht prtsent invontion may be
native peptides present in living body (endogenous
pept~.des). ~, typical rP-10 according to the present
invention is generated by a sequence encoded by SEQ ID NO:
~. to be generated, for example, polypeptides having an
am~.no acid sequence shown by SEQ ID NO: 2 or ~.ts
essentially equ.fvr~lent amino acid sequences. A typical zP-
according to the present znventxon includes at least 1
to 102 continuous amino acid residue of the amino acid
CA 02497497 2005-03-02
- 23 -
sequence shown by SEQ ID NO: 2, and has a biolog,iaal
~tGtivity selected from the group consisting of (a)
activating concoptus migration, {b) promotj.ng conceptus
implantation on the uterine wall, (c) treating sterility,
(d) promoting pregnancy, (e) corit~tvlling interaction
between conceptus and mat~rnal system, (fi) activating
immunocyte migration, and (g) Controlling immune function
in the uterus. furthermore, zP-10 according to the present
invent~,ot~ includes a polypeptide exhibiting characteristics
as in above and having a homology to the amino acid
sequence shown by sEQ ID NO: 2 at least 60~b, preferab~.y at
least 75.5~b, stiZ~. mare preferably at least 82.7R~, even
more preferably at least 90~, and most preferably at least
95~. Novel one is the specifically preferable.
Ext~mples o~ "polypeptide" according to the
present invention include TP--10, and relating polypeptides
thereof, especially ungu7.ate xP-lOs such as ovine IP--10 and
caprine IE-10. As for pregnancy, the zp-zo arsd its
relating polypeptides may be derived from hums~n from the
viewpoint of using ate activity during implantation.
Examples of the polypepta.de znclude ovine zP-10, typically,
peptides having a homology with an amino acid sequence
shown by SEQ TD NO: 2 at least 60~, preferably at least 70$,
still more preferably at least 80Rs, even more preferably at
least 85Rs, even more preferably at least 90$, evmn more
preferably 95$, and most preferably at least 97~, and
having a biological activity selected from the group
ca~sisting of (a) activating conceptus migration, (b)
promoting conceptus implantation on the utexxne wall, (c)
treating sterility, (d) promoting pregnancy, (e)
controlling interaction betr~teen concoptus and maternatl
system, {f) activating immunocyte migration, and (g)
controlling immune function in the uterus, or substantially
equivalent biological activity such as antigenicity.
CA 02497497 2005-03-02
-24-
IP-10s according to the present invcnt~.pn include
zE-10 molecules derived from human and ungulates such as
ov~,ne and goat, and novel amino acid sequences oath having
a distinctive domain or motif or a segment thereof w~,thxri
IP-10, from the viewpoint of using an activity during
implantation for pregnancy. Moxe pxeterably, peptides
according to the present invention include amino acid
sequences having an activity during implantation for
pregnancy described above, and hava.ng a homology with each
IP-10 fam~.~.y at least 60~b. Typically, the pepta.de
according to the present invention a.s selected from the
group consisting of peptides containing an am3.no x~cid
sequence substantially equ:cvalent to the amino acid
sequence shown by SEQ xD NO: 2. Furthermore, the peptide
according to the present invention ~.ncXudes a part or whole
amino acid sequence showr~ by SEQ Z~ NO: 2. These sequences
axe included in the prESent invention.
The term "homo7.ogous" or "homology" as used
herei,r~ means the amount (or number) of amino acid residues
or bases, which are identified as the same with a~xxothex
amino acid xes~.dues or bases from polypeptide sequence (or
amino acid sequence) or polynucleotide sequence (or
nucleotide sequence), respectively, in the reJ.ation of
compat~.bi7.ity between the two chains, i.e. a degree of
sequence correlation in two po7.ypeptxde sequences or in two
polynucleotide sequences. I3omology can be easily
calculated. Many methods for measuring a homology between
two polynucleotide sequences ox po~.ypeptide sequonces are
known. A term of homology (or identity) is widely known to
those skilled in the art. (For example, Lesk, A. M. (Ed.),
(1988) Connputational Molecular Biology, Oxford University
Press, New York: Smith, D. W. (Ed.), (1993) Siocomputirrg:
Znfoxmatics and Genome Erojects, Academic Press, New York;
Grifin, A. M. & Gritin, H. G. (Ed.), 0.994) Computex
Analysis of Sequence Data: Part T, Human Press, New ,Terseyt
von ~ieinje, G~, (1987) Sequence Analysis in Molecular
CA 02497497 2005-03-02
-zs-
Biology, Academic Press, New York: Gribskov, M. & Devereux,
J. (Ed.), (1992) Sequence Analysis Primer, M-Stockton Press,
New xoxk). The general methods for detexmining a homology
between two sequences axe, but not limited to, disclosed in
Martin, J. Bishop (Ed.), (1.990 Guide to Huge Computers,
Academic press, San Diego; Carillo, H. & i~ipmaa, b., (1988)
SIAM J_ Applied Math., 48: 10?3, etc. Preferable methods
for determining a homology are designed to identify the
most aompz~tible parts in two sequences o.f a question.
These methods may be assembled in a computer program.
ExampJ.es of the preferable computer program fox determining
a homology between two sequences include, but not limited
to. GCG program package tDeworeux. J. et al., (~.98~)
Nucl.sic Acids Research, 12(1): 38?), BhAaTP, BI~SxN, and
FASTA (Atschul,, S. P. et al-, (1990) J. Molec. Biol., 215:
403). N,fethads widely known in the art can be also used.
A nucleic said ericading each polypeptide ar
protein according to the present invention, fxom the
viewpoint og using the activity during implantation for
pregnatxcy described above, may include within the sequence
a nucleotide sequence encoding a member selected from TP-10,
rP-10-relating polypeptidesr or their segments having
continuous amino acid xesidues: ar a nucleotide sequence
encoding a member selected from IP--1.0 dexived f7COm
ungulates such as ovine and c~vat, its relating polypeptides,
IP-10 derived from human, its relating polypeptides, or
their continuous amino acid sequence segments . ,i~. typ7.cal.
nucleic acid contains a nucleotide sequence encoding the
peptide shown by SEQ ID NO: 2 or its segments having
continuous amino acid residues, for example, a nucleotide
sequence composed of at least positions 63--365 of the
nucleotide sequence shown by SEQ rD Nc3: l, d nucleotide
sequence composed of from ATG of 60-62 positions to TAA of
positions 366-368 of the nucleotide sequence shown by SEQ
ID NO: 1 (the termination codon TAA may be TGA or TAG), and
a nucleotide sequence added an initiation codon (i.e. codon
CA 02497497 2005-03-02
~ncoding Met) and a termination codon. Furthermore, any
nuc~,ei.c said cart be included as long as the nucleic acid
containing a lxuGleotide sequence equivalent to the above
nucleotide sequences, for exampXe, z~ nucleic acid
containing a nucleotide sequence e:'7~coding a peptide
conta~.n~,r~g ari amino acid sequence having a homoxogy with
the proteins encoded by the above-mentioned nucleotide
sequences at least 80~b, 85~, 905, 955, or 98~, and
containing at laz~st 1 to ~.6 continuous amino acid xesidues
of the amino acid sequsnce shown by SEQ rD NO: 2, and also
having a biological activity selected from the group
consisting of (a) act~,vating conceptus migration, (b)
promoting conceptus implantation on the uterine wall, (c)
treating sterili'~y, (d) promoting pregnancy, (e)
controlling interaction between conceptus and maternal
system, (f) activating immunocyte migrat,i.on, and (g)
controlling immune function ~.n the uterus, or a~
substantially equivalent biological activity such as
antigenic3.ty. Examples of the nucle~.c acid enCOding TE-~.0
acre single-stranded DNA, double-stranded DNA, RNA, DNA: RNA
hybrid, synthetic DNA, and the like. The nucleic acid may
be an ovine, caprine, ox human c~enomia DNA: an ovine,
capxxne, or human genomic DNA library: cDNA derived from
ovine or human tissues (or sells): or a synthetic DNA. The
nucleotide sequence of the nucleic acid encoding IP-10 may
be modified (tor examp7.e, addition, deletion, and
subst~,tution). These modified nucleotide sequences are
also included in the present invention. The nuc7,eic acid
accoxd~.nc~ to the present invention may encode a peptide or
its segment of the present invention as described below,
and DNA is pxefexable. The tez~m "nucleotide sequence
equivalent" means, for exarnpJ.e, a nucleotide sequence
capable of hybridizing with a continuous at least 5
nucleotide sequences, preferably at least 10, more
preferably at least 15, still morn preferably at least 20
nucleotide sequencQ in the nucleotide sequence shown SEQ ID
NO: 1 under a stringent condition, and substantially
CA 02497497 2005-03-02
_27_
equ~.v$lent to ZP-Z0. The stringent condition is a
condition enabling seloGtively detectable specif~.e biridirig
of a predetermined polynucleotide with ars oligonucleotide
probe ox a polyt2ucleotide probe. The stxa,rigerit GOridition
is defined by a salt Content, an organic solvent (for
example, formamide), temperature, and other known
condit~.ons. Nrunely, the stringency increases by decreasing
a salt content, increasing an organic solvent cor~terit, or
raising a hybridization temperature. For example, a
stringent salt aoritent is generally lower than about 750 mM
NaCl and lower than about 75 mM sodium citrate, more
prefESably, lower than about 500 mM NaCl and lower than
about 50 mM sodium ci'Crate, and most preferably, lower than
about 250 mM NaCl and lower than about 25mM sodium citrate.
A stringent oxgan~.c solvent content ~.s higher than about
35Rs formamxde, mast preferably, higher than about 50R~. A
stringent temperature is h~.gher than about 30°C, more
prefdrably, higher than about 37°C, and most preferably,
higher than about 42~C. Other conditions are a
hybridization time, a washing agent (fox example,
surfactant such as SbS) content., and the presence of a
carrier DNA, and the like. Combination of these conditions
defines various stringent conditions. Fox example, a
condition defined by a sodium content of about ~.9 to 20 mM
and a temperature of about 60°C to 65°C is preferable, mere
preferably, a sod~.um cori,tent of about 19 mM and a
temperature of about 65°C.
The confi.xmatior~ of predetermined gene products
may be performed by using appropriate animal cells such as
293T cell and COS--1 ce7.7., which axe trarisfected with the
genes. The introduction of the foreign genes to animal
cells such as mammal cells can be pex~orttted by methods
knowtl in the field of the art or by substantially
equivalent methods thereof, such as a calcium-phosphate
method (for example, F. L. Graham et al.,(1973) Virology,
5~: 456), a D~AE-dextrari method (for example, D. Warden et
CA 02497497 2005-03-02
-28-
al.,(1968) J. Gen. Virol., 3: 371), an electroporation
method (fox example, E. Neumann et al., (1982} EM$O J, 1:
841), a micro-inject~.on rrtethod, a liposome method, a viral
infection method, and a phage particle method. Gene
products produced by animal cells transfected with a
predetermined gene, for examp~,e, Zp-10 gene can be analyzed.
Any plasmid can be used as a plasmid
incorporat~.ng DNA prepared according to the present
invention (for example, IP-10 gene) as long as the plasmid
can express the DNA xn ~x host cell (for example,
prokaryot~s such as Escherichia coli and $acillus subtilis,
yeast, eukaryotic cells such as CHO ce7.~. and COS cell, and
insect cells such as sf21) widely used in a genetic
engin$ering field. Such sequences may contain, for example,
a modx~xed cadon appropriates for the expression in a
selected host cell, a restriction enzyme moiety, a
controlling or promoting sequence far the readily
express~.on o~ a target gene, a linker and adaptor for
binding a target gene, and also useful sequences (incyud~.ng
genes encoding hybrid proteins and fused proteins) for the
control oi° antibiotics or the ~.xke, rnetabalism, and
selection. Preferably, appropriate promoters axe used.
For example, plasmi.ds usxzlg Escherichia coli as a host cell
may use a tryptophan promoter (trp), a lactose promoter
(lac), a txyptophan-lactose promoter (tae), a lipoprotein
promoter (~.pp), 1.-phage PL promoter, etc., plasmids using
animal cells as a host cell may use SV40 late promoter,
MMTV LTR promoter, RSV LTR promoter, CMV promoter, SR ~
promoter, etc., and p3.asm~.ds us~.ng yeast as a host cell may
use GALL, GAL10 promoters, etc.
Examples of the plasmid us~.ng EschexxGh~.a aali as
a host cell include p81t322, pUCl8, pUC~.9, pUC~.IB, pUC119,
p5P6A, pSP65, pTZ-1$R/-7.8U, pT2-19R/-7.9U, pGEM-3, pGEM-~1,
pGEM-32, pGEM-4Z, pGEM-5Zf(-), and pEluescript KSxM
(Stratagane). Examples of a plasmid vector suitable for
expression in Escherichia coli include pAS, pKK223
CA 02497497 2005-03-02
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(Pharmaaia), pMC1403, pMC931, pKC30, and pRSET-$
(Tnvitrogen). Examples o~ the plasmids using animal cells
as host cells include SV40 vector, polyoma virus vectors,
vaccinia virus vectors, retrovirus vectors, for example,
paD, pcD-SR a, CDM8, pCEV4, pMEl8S, p$C~.2$z, and pSG5
(Stratagene). Examples of the plasmid us~.ng yeast as a
host cell include Yip type vectors, YEp type vectors, YRp
type vectoxs, and YCp type. vectors, for example, pGPC-2.
As for the host cells, examples of Escherichia coli as the
host cell a.nclude Esaherichia coli K12 strain-derived
strain such as NM533, XLl-Blue, C600, DH1, DHS, DH11S,
DH12S, DHSa, nH208, HB101, MC106~., JML09, and ST8L2, and
8834 strain-derived strain such as BL27.(DE3)pLy~xS.
Examples of the an~.mal cells as the host cell include
African green monkey ,f~.bxc7blaSt-derived COS-7 cells, COS-I
cells, CV-Z Cells, mouse fibroblast-derived COp cexls, MOP
cells, WOP cells, Chinese hamster cell-dmr3ved CHO cells,
CHO DH FR- cells, human ~IE~A cells, marine cell--derived C127
cells, and marine cell-derived NTH 3T3 cells. Regard~.ng
the insect cells, Bombyx mori nuclear polyhedsosis virus or
a derivative thereof may be used as a vector, and silkworm
larvae or cultuxed s~.~.kwoxm cells such as BM-N cells may be
used. It is also possible to use plant cells as host cells,
the plant cells axe widely known in the field of the art as
well as appropriate vectors. Zn the genetic engineering
technique accoxd~.ng to the present invention, restriction
enzymes, reverse transcriptase, DNA modifying or
decomposing enzymes for modifying ox convextj.ng DNA
fragments to structures suitable for cloning, DNA
polymerases, terminal nucleotidyl transferases, and DNA
ligasas can be used. Examples of vectors suitable for DNA
gene clon~.ng to conatruat DNA libraries include plasmids, ~.
phages, cosmids, P1 pharges, F faatox, and YAC, preferably,
the ~, phage-derived vectoxs such as Chaxozx 4A, Charan 21A,
A gtl0, ~l gtll, ~. DASHTT, h FTXTT, ~. EMBL3, and ~. ZAPIITM
(Stratagene).
CA 02497497 2005-03-02
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A transioxmant asccording to the present invention
is obtained by transformation with a,n expression vector
containing a nuoleic acid encoding a prote~.t~ of the present
imrention and a cell line of the transformant having high
and stable expression capability can be obtained by
repeating cloning optxanally using an appropriate selective
maxker. For example, when the dhfr gene is utilised as the
se7.ective marker in transformant cells derived from animal
cells as host ce~.~.s, a cell line, which amp7.z~ies a DNA
encoding the protein of the present invention and causes a
higher level of expression, can be obtained by culturing as
gradually inaxeasing the MTX concentration, and then by
selecting .a resistant strain. 9Phen the transformant of the
present invention is cultured under conditions enabling the
expxes~s3on of a nucleic acid encoding the protein of the
pres~nt invention, the txansformant can produce and
accumulate the target product. The transfoxmant can be
cultured in a medium that is coituttpnly used in the art. For
example, a liquid medium can be suitably used for a
transformant derived fxom a prokaryotic cell such as
Escherichia coli and Bacillus subtilis or Pram yeast as a
host cell. The medium can contain a carbon source, a
nitrogen source, inorganic substances, and so forth which
are necessa,xy for the growth of the transtoxmant. Examples
of the carbon source ~.nalude glucose, dextrin, soluble
starch, and sucrose. Examples of the nitrogen source
include inorgan~.c ax organic substances such as ammonium
salts, nitrate Salts, corn steep liquor, peptone, casein,
meat extxaGt, malt. extract, soybean cake, potato extxaGt.
The inorganic substances inc~.ude calcium chloride, sodium
dihydrogenphosphate, magnesium chloride, and calcium
carbonate. Yeast, vitamins, casamino acids, growth
promoters, and the like may be also added. An agent such
as 3/3-~,ndolylacrylic acid is optionally added so that the
promoter can efficiently function. Preferably, the medium
has a pH of about 5 to g.
CA 02497497 2005-03-02
-31-
lrt the vase of Escherichia coli, the culture is
gonera~lly performed at about 15°C to X35°C for about 3 to 75
hours. Aeration and stirring axe performed if necessary.
In the culture of a transformant derived from anima~~. cells
as host cells, a medium such ass MEM medium containing about
5'b to 20~ fetal bovine serum, PRMZ 1640 medium, or DMEM
medium is used. The pH is preferably about 6 to 8. The
culture is genexa~.~.y performed at about 30°C to 40°C for
about ~.5 to 72 hours. If necessary, aeration and stirring
are performed. zn the extraction from the ce~,7.s cultured
as ~.n r~bovm~, the bacteria or cells are collected by a
conventional method after the culture. For example, the
collected bactexXa or cells are suspended in nn appropriate
buxfer solution and then disrupted by, for example,
sonication, lysozyme, and/or freezing and thawing. Then, a
crude extract is prepared by centrifugation or filtration.
A protein denaturing agent such as urea and guanidine
hydroch~.oxide, and a surfactant such as Tx~.torx x-100
(trademark) and Tween-$0 (trademark) may be added to the
bu~far solution. When the target product is secreted into
the culture fluid, the supernatant is collected by
srptsration from the bacteria or ce7.J.s by a per se known
method after the completion of the culture. The target
product in the resulting culture supernatant or extta~ct can
be purified by an appropriate combination of per se known
methods of sepa~ra~tion and purification. The product can be
purified by, for example, salting out with ammonium sulfate,
gel filtration using Sephadex, ion exchange chromatography
using a carrier having diethylam~,noethyl or carboxymethyl
groups, hydrophobia chromatography using a carrier having
hydrophobic groups such as butyl, octy~., ox phenyl groups,
dye gel chromatography, electrophoresis, d~.a~,ysxs,
ultratiltration, affinity chromatography, and high-
performance liquid chromatography. Preferably, the product
can be pux~.f~,ed ,and isolated by the treatment with
polyacrylamide gel electrophoxesxs, or affinity
chromatography utilizing an immobilized ligand or the like.
CA 02497497 2005-03-02
-32~
For example, gelatin--agarose affinity chromatography and
heparin-agarose chromatography may be used.
Furthermore, modified proteins corresponding to
IP-10 (for example, human IP-10, ovine IP-7.0, and capx~.ne
IP-10) as xesuJ.t~.ng from mutation such as substitution,
deletion, insertion, translocation and addition of one or a
plurality of amino acids of the TP--10 can be produced based
on the nucleotide sequence if the gene according to the
present invention by using a method genexaXl.y used xn
genetic engineering. As the methods of such mutation,
conversion, or modigication are descx~,bed iri Japanese
B~.ochemical Society (ed.), (1986) "Zoku Seikagaku Jikken
Koza (Experiments in Biochemistry, Second Series) l, Ide~nsi
Kenkyuho (Methods of Gene Studies) II", p. 7.05 (Susumu
Hixose), Tokyo Kagaku Dozin; JapanesE Biochemical Society
(ed.), (1992) "Shin Seikagaku Jikken Koza (Experiments in
Biochemistry, Updated) 2, Kakusan (Nucleic Acids) III
(Recombinant DNR Technology", p. 233 (Susumu Hirose), Tokyo
Kagaku Dozin: R. Wu, L. Grossman, ed., (1987) "Methods in
Enzymology", Vol. 154, p. 350 & p. 367, Academic 8xess, New
Yoskp R. Wu, L. Grossman, ed., (1983) "Methods in
Enzymol.ogy", Vol. 100, p. 457 & p. 468, Academia Press, New
York: J. A. Wells, et al., (1985) Gene, 34: 315: T.
Grundstsoem, dt al., (1985) Nucleic Acids Res., 13: 3305; J.
Taylor, et al., (1985) Nucleic Acids Res., 13: 8765; R. Wu,
ed., (7.987) "Methods in Enzymology", VOl. 155, p. 568,
Academic Ersss, New York; A. R. Oliphant, et al., (1986)
Gene, 44: x.77- Far example, there may be mentioned methods
such as site-directed mutagenesis (site~specific
mutagenesia) using a synthetic aliganucleatide (taller, et
al. , ( 1987 ) Nucl . Ac~.ds rtes. , 10 : 6Q87; Carter, et a7.. ,
(1986) Nucl. Acids Res., 13: 4331), Cassette mutagenesis
(We~.ls, et al. , 0.985 ) Gene, 34 : 3~,5 ) , Restxa.ction
selection mutagenesis (Wells, et al., (1986) Philos. Trans.
R. Soc. London Ser A, 37.7: 47.5 ) , alani.ne scanning
(CUnningham & Wells, (1989) Science, 244: 1081-1085), ECR
CA 02497497 2005-03-02
-33..
mutagenesis, Kunkel method, dNTP[a S] method (Eckstein), and
region-directed mutagenesis using sul.fuxaus acid or nitrous
acid.
Further, the resulting proteins (or peptides)
according to the present invention can be modified by atmino
said residue contained therein by a chemicaX technique, ar
can be a~.tere~d into its derivatives by modification or
partial decomposition by enzymes such as peptidases, ~.g.,
pepsin, chymotrypsin, papain, bromela~.n, endapeptidase, and
Qxopeptidase. In general, the protein according to the
present invention has a aaxboxyl group (-COOH) or a
carboxylate group (-COO-) at the C-terminal, but may have
an amido group (--CONH2) or an ester (--COOR) at the C-
terminal. The A xn the ester may be a C~-g alkyl group such
as methyl, ethyl, n-propyl, isopropyl, and n-butyl, a C~_8
cycloalkyl group such as cyclvpentyl and cycloalkyl, a
aryl gxoup such as phenyl and a,-naphthyl, or a C~-xq aralkyl
gxoup including a phenyl-C~-Z alkyl group such as benzyl and
phenethyl, and an a -naphthyl-C~_Z alkyl group such es a -
naphthylmethyl, as well as a pivalvylvxymethyl group which
is widely used as an oral administration ester. L~lhen the
protein according to the present inventic~ri has a caxboxy
group (ox Caxboxylate) other than at C-terminal, the
carboxy group may be am~.dated yr esterified. Such proteins
axe also included in the present invention. Such esters
are the same as the eatex at C-terminal described above.
Fuxthermvxe, examples of the above-mentioned
proteins according to the present invention include
proteins of which meth~.on~.ne xesidue at the N-terminal. is
protected by, for example, a CZ_6 acyl group such as a C2_5
alkyl-carbonyl group, m.g. formyl and acetyl: proteins
having pyroglutamated N-terminal which is converted from a
glutamyl group generated by breakage o~ N-terminal in
living body, pxotexns of which a substitute (e.g. -4H,
-COON, amino group, imidazole group, indol group, guanidine
CA 02497497 2005-03-02
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group) on a side chain is protected by a protecting group
(i.e. Cl-6 acyl gxoup such as formyl, acetyl), or proteins
on which a sugax chain is bound, i.e. conjugated protein
such as glycoprotein. Tn producing by gone recombinant
technology, i~C is possible to cause expxess~.on o,f Ip-10 in
the form of a fused pxotein. The resulting IP-~.0 may be
changed ox processed in vivo or in vitro into a protean
having substentxa~~.ly the same biological activity as native
IP-10. The fusion protein can ba produced by a gene-
engineexing method widely used, and can be pux~.fied by
affinity chromatography utilizing the fused portion thereof.
Examples of the fused pxotein include proteins fused to
histidine tag, ox j~~goloctosidase ((3-gal), maltose--binding
protein (MBE), glutathione-S-transferase (GST), thioradoxin
(TRX), ox cxe xeaombinasa amino acid sequence. Similarly,
a heterogeneous epitope tag is added to the polypeptxde for
enabling purification by x~rutwnoaf~inity chromatography
using an antibody specifically binding to the epitope. =n
a more suited embod~.ment of the epitope, AUS, c--Myc,
C7euzTag 09, CruzTag 22, CruzTag 41, Glu~Glu, HA, Hall, KT3,
FLAG (registered trademark, sigma-RJ.drich), Omni-probe, S-
probG, T7, Lex A, V5, VP16, GAL4, and VsV-G axe rneritXOned
as examples. See, F~.e7.d et al . , ( 1988 ) Molecular and
Ce~.~.ulax Biology, 8: 2159-2165; Evan et al., (1985)
Molecular and Ce~.~,ulax Biology, 5: 3610-3616; Paborsky et
al., 0,990) Protein Engineering, 3(6): 547-553: Hopp et al.,
(1988) BioTechnology, 6: 1204-127.0; Martin at al., (1992)
Science, 255: 7.92-194; Skinner et al., (1991) J. Biol.
Chem., 266: 15163-151661 Lutz-Pxeya~uth et al., Proc. Natl.
Aced. Sci. USA, 87: 6393-6397 (1990); etc.).
Further, the fused protein may be labeled with a
marker to be detectable. In a more prefexxed embodiment,
the detectable marker may be Biotin Avi tag based on the
biotin/streptavidin, a fluorescent substance, end so Earth.
Examples of the fluorescent substance include green
fluorescent pxotein (GfP) derived from fluorescent
jellyfish such as Aequorea vietorea; var~.ants thexeof (GFP
CA 02497497 2005-03-02
- 3S -
variants) such as Enhanced-humanized GfP (EGFP), red-shift
GFP (rsGFP), yellow fluorescent protein (YFP), green
fluorescent protein (GFP), cyan fluorescent protein (CFP),
and blue fluorescent protein ($Fp) ; and GFP de~:~.ved fxom
Renilla reniformis (Atsushi Miyasawa (ed.), (Z000) J~,kkeri
Tgaku (Experimental Medicine), an extra number,
Posutogenome Jidai no Jikken Kaza (Experimonts in
Postgenomic Era) 3-GFp and BxoxmagXng, Xodosha). xhe
detection can be also performed using an antibody
(including a monoclonal antibody and a fxagment thexeo~)
capable of specifically recognizing the fused tag. The
two-hybrid method using yeast can be also utilized.
Examples of the object proteins (for example,
human IP,10 and ovine IP-7.0) may include a pxotein herring
at least one amino acid residue being different from that
of a native protein in the terms o~ identity and position,
and a protein having a specific activity during
implantation, for example, an activity during implantation
far pregnancy, more specifically, a biological activity
selected from the gxoup consisting of (a) activa~txng
conceptus migration, (b) promoting conceptus implantation
an the uterine wall, (c) treating sterility, (d) promoting
pregnancy, (e) controlling interaction between conceptus
and maternal system, (f) activating immunocyte migration,
and (g) controlling immune function in the uterus.
ExampJ.es of the object protein of the present intrention
include deletion analogues lacking one or more (e.g., 1 to
80, preferably 1 to 60, more preferably 1 to 40, further
preferably 1 to 20, particularly 1 to 10) amino acid
residues peculiar to the human TP-10 or o~stine TP-10,
substitution analogues having one ox moxe (e.g., 1 to 80,
preferably 1 to 60, more preferably 1 to 40, further
preferably 1 to 20, paxticuZarly 1 to ~.0) pecu,liar amino
~soid residues substituted by other residues, and addition
analogues having one or more (e.g., 1 to 80, preferably 1
to 60, moxe pxe~arably 1 to 40, further pre:ferab~.y ~, to 20,
CA 02497497 2005-03-02
particularly 1 to 10) amino ac~.d xesidues added thereto.
The present invention encompasses all of the mutants such
as those described above as long as they maintain a domain
structure or receptor binding activity which is
chaxaateristics of native IP-10. Furthermore, the present
invention may encompass tl-xose having a primary structure
corifoxmation substantially equivalent to that of native r8-
~.0 or a part thereof, and also those having a bioXogi.csl
activity subatanti~xlly equivalent to that of native IP-10.
The object proteins of the pxesent invet'ition have a
homology of at leas' 60~, preferably higher than 70~, more
preferably highex than 80~ or 90$ with, Eor example, an
amino ac~,d sequences selected from the group consisting of
(~.) amino acid residues from 2- to 102-positions, (2) from
1- to 102- positions, arid (3) from 20- to BO-positions of
amino acid shown by sECa No: 2. A paxt of the abject
protein of the pxesent invention means a partial peptide of
these proteins (i.e. partial pept~.de of tha protein)
show~,ng s~ubs~tantially equivalent activities with ovine I~-
y0 according to the present invention. For example, the
partial peptide txccordit7g to the present invention has at
least 5 amino acids residue, pxefexably at least 20 amino
acid residues, more pxefexably at least 70 amino acid
residues, fuxtZxex preferably at least 80 amino acid
residues, furthex preferably at least 90 amino acid
residues, particularly apt least 9."a amino acf.d xes~.dues of
ovine IP-~.0 structure amino ac~.d according to the present
invention. Qreferably, these amino acid residues axe
continuous, or axe the same as the corresponding portion of
the amino acid shown by SEA ID NO: 2 in the teams of
homology.
In the present specification, the terms
"substantially equivalent" and "substantial.l,y the same"
mean that the activities of a protein, for example its
activity during implantation pexiod, physiological activity,
and biologxaal activity, are substantially the same as
CA 02497497 2005-03-02
-37~
those of another protein. xhe terms include, within the
meaning thereof, the case of a protein having a
substantiaX~.y the same nature as compared with another.
The activity of substantially the same nature ~.a, for
exampl~, a biological activity selected fxom the group
consist~.ng of (a) activating conceptus migration, (b)
promoting conceptus implantation on the utexine walls (c)
treating sterility, (d) promoting pregnancy, (e)
controlling ~.nteraction betwten concmptus and maternal
system, (f) activating immunocyte migration, and (g)
controlling immune function in the uterus. The activities
of substantially the same nature indicate that the
activities are each qualitatively the same nature, for
example physiologically, pharmacologically, or biologically,
as compared with the countexpaxt. Fox e~camp~.e, xt is
preterxed that the activities selected from the group
Consisting of (a) activating conceptus migration, (b)
promoting conceptus implantation on the uterine wall., (c)
treating sterility, (d) promoting pregnancy, (e)
controlling interaction between conceptus and maternal
system, (f) activating immunocyte migration, and (g)
controlling immune funetxon iri the uterus, be equivalent
(for example, about 0.001 to about 1,000 times, preferably
about 0.01 to about 100 times, more preferably about 0.1 to
z~bout 20 times, further preferably about 0.8 to about 2
times) to the counterpart. Howevex, the l,evel.s of thes~
act~.v~.t~,e~s, they molecular weight of the pretein, and other
quantitative factors may differ. Substitutions deletion,
or addition of amino acid may not often modify they
physiological or chemical characteristics, but may
advant~xgeously modify them. zn such cases, a polypeptide
app~.xed the substitution, deletion, or adaitian may be
substantially the same as those being not applied such
subst~,tution, deletion, or addition from the terms of
predetermined objects. Examples of the substantially the
same substitu~ent in the amino acid sequence can be selected
from other amino acids ~.n the gxoup [fox example, nonpolar
CA 02497497 2005-03-02
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(hydrophobic} amino acids such as alani.ne, phenylalanine,
leuc~.ne, isaleucine, valine, proline, triptophan, and
methionine: polar (neutxalj amine acids such as glycine,
sexxne, threonine, cysteine, tyrosine, asparagine, and
glutamine: positive charg~d amino acids (basic amino acid)
such as arginine, lysine, and hxstidine: and negative
charged amino acids (acidic amino acid) such as aspaxtic
acid and glutamic acid], to which the substituent belongs.
For synthesizing the protein arid partial peptides
thereof according to the present invention, methods known
in the field of peptide synthesis, fox examp~.e chemical
synthetic method such as a liquid phase method and a solid
phase method cari be used. zn such methods, peptides are
synthesized by b~.nding adequately protected amino acids one
by one on a resin used in protein ox peptide synthesis to
form a des7~red amino acid sequence by any of various per se
known condensation methods. Regard~.ng the condensation
reaction, carbodiimides such as dicyclohexylcarbodiim~.de
x~re preferable r~agents. When the resulting product has a
protective group, removal of the protective group is
appropriately perfo~cmed to yield the target product.
Regarding the protein and its partial peptides of
the present invention, when they are obtained in the free
form, they may be converted to salts by a per se known
process or a modification thereof. When they are obtained
in a salt form, they may be converted to the free form or
to other salts by a per se known method or a modification
thereof.
The salts of the protein and partial pept~.des
thereof according to the present ~.nventi.on preferably
include, but are not limited to, physioxog~.cally ar
pharmaceutically acceptable ones. Such salts may include
salts with inorganic acids such as hydroch7,aric acid,
hydrobromic ac~.d, sulfuric acid, nitric acid, and
phosphoric acid: and salts with organic acid such as acetic
acid, formic acid, malefic acid, fumaric acid, succinic ac~.d,
CA 02497497 2005-03-02
-39-
citric acid, tartaric acid, malic acid, benzoic acid,
methanesulfonic acid, p-toJ.uenesulfonic acid, and benzene
suJ.fonic said. The salts may also include ammonium salts
and salts with organic bases such as ethylamine,
dimethylamine, tximethylamine, and hydoxyethylamine.
These IP-1b, its mutants, modifications, and
derivatives cart be separated and purified by the above-
mentianed methods. In the present invention, the terms
"frx~grnerit", "derivative", and "analogue" mean the
polypeptide that essent~,aJ.ly retains the same biological
functions or activities of the polypeptide showrs by S~Q ZD:
2, the polypeptide that is encoded by an hnRNA or mR.NA thaw
are transcribed from the sequence shown by SEQ ID NO: 1 and
received or not received specific splicing, ox the
po~.ypeptide encoded by genomic DNA, when the terms
"fragment", "derivative", and "anaJ.ogue" are used in
connection with these poptides.
Since the pr~sent invention provides unknown
information reXating to functions of xP-10 protein derived
mammal, the utilization of such information is inc~.uded in
the present invention. Such utilization includes the
development of screening methods ox xeagentx for
identifying the functions of the zE-Z0, for ~xample,
activating conceptus migration, promoting conceptus
implantation on the uterine wall, treating sterility,
promoting pregnancy, activating conceptus migration to
uterus during early pregnancy, and controlling interaction
between conceptus and maternal system, by isolating and
detecting genornic Di'TI~ arid aDNA derived from mammal,
especially human, encoding the TF-1Q, and related proteins
thereof.
For example, by using an zP-~.0 l~N~r. sequence such
as ovine xP-10 DNA sequence as a probe, a function analysis
of IP-10 and its xel.ated proteins, fdr example activating
conceptus migration, promoting conceptus irnp7.antati,on vn
the uterine wall, treating sterility, promoting pregnancy,
CA 02497497 2005-03-02
-40-
activating conceptus migration to utexus de~xxng early
pregnancy, and controlling interaction between conceptus
and matexnal system, can be performed. The probe is
optionally labeled with a label known in the field of the
axt. The label includes radioisotope (RI) and non-RT, and
the non-RT is preferable. ExampJ.e~c of the non-RZ label
include fluorescence label, biotin label, and chemical
luminescence label. The fluorescence label is most
prefexabZe. fluorescence that can bind with base portion
of an oligonueleotide is suitably selected. Examples of
the fJ.uorescence libel include cyanin~ dyes (e.g. Cy3 and
Cy5 belonging to Cy nyeTM sexies), xhodamine fiG reagent, N-
acetyl-NZ-acetylaminofluorene (AAF), snf AAIF (iodide
derivative of AAF). For example, the isolation of gene can
be performed by ECR and RT-PCR (i.e. PCR using reverse
transcriptase). zP-10 cDNA ahd its xelated DNAs can be
used for isolation and detection of IP-10 related gene by
PCR, competitive pCR, Rf-fCR, Real-x5.me fCR (Journal of
Molecular Endocrinology, (2000) 25, 169-193), and other
methods using a DNA primer (including a primex set). The
pxaxner is designed according to a specific sequence region
selected from an amino acid sequence deduced ~rom cloned
x1~-J.0 cD~l,~ se~c~uence and chemically synthesized. The primer
size (the number of base) is 15 to 90 bases, preferably 15
to 30 bases, for specific annealing with a template DNA.
Long accurate (7~A) PCR requires at least 30 bases. A pair
of (two) primers consisting of sense strand (5' side) and
antisense strand (3' side) is avoided to have a sequence
complementary to each other so as not to anneal with each
other, as well as avoided to have a self~complementary
sequence sv as nvt to form a hairpin structure within the
primers. Furthexmore, the primers should contain GC at a
contmnt of about 50~ in order to sQCUre a stable binding
with the tempXate DNA, and should be avoided to have GC-
rich or AT-rich regions. Since the annea~linc~ temperature
depends on the melting tempex'atuxe (Tm), the primexs having
approximate Tm values ranging from 55°C to 65°C should be
CA 02497497 2005-03-02
-41 -
salacted in order to obtain h~.gh~.y specific PCR products.
Furthermore, it is required to adjust the primer final
concentration in PCR to about 0.1 to llxM. Commercially
available software for designing primers, fox example,
OligoTM (Nati.onal Bioscienc~ Inc., U.S.A.), GENETXX
(Software D~v~lopment Co., Ltd., ,?span) can be used.
Universal, primers, i.e. known primers such as dT primer can
be used. For examplQ, IP-10 mRNA expression in human
tissues can be studied by Northern blot analysis for poly
(A)' RNA derived from various tissues. According to the
present invention, IL~-10 mRNA expression in human tissues,
rP-l~ gene itself, and so ,forth can bQ detected or measured
by using the cDNA as a probe by, for example, Northern
blotting, Southern blotting, ~.n situ hybridization, dot
blotting, and RNase protection assay. Consequently,
cellular protein metabolism in cells, activation of hormone
precursor, and physiological functions involved xwsrarious
pxoGesses in normal cells including the above-mentioned
activities xdlz~ting to pregnancy such as a role ~.n
interaction between conceptus and mother, sterility, and
abortion can be performed. The cDNA can be also used for
gene diagnosis of diseases relating to IP-10. The
abnormality in nucleic acids encoding the IP-7.0 and its
related proteins, for example, dxxmage, mutation, and a
decrease or increa~sm in expression can be diagnosed by such
gene diagnosis.
In situ hybridization includes direct and
indirect non--RZ in situ hybridization. In the direct
method, a detectable molecule (reporter) is directly bound
to the nucleic acid probe, and in the indirect method, for
example, an antibody against the reporter molecule a.s used
to amplify the s~.gnal. A functional group (for example, a
primary aliphatic amino group, an SH-gxpup) is introduced
to oligonucl.eot~.de in the nucleic acid probe, and may bind
to a hapten, a fluorescent dye, or an enzyme. Extsmples of
the label of the nucleic acid include digoxigenin (DIG),
biotin, and fluoresceine, and can be appropriately selected
CA 02497497 2005-03-02
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from labels described blow in a description of antibodies.
Furthermore, multilabex7.~.ng can be used, and a labeled
antibody can be also used. Regarding the labeling~of the
nucleic acid probe, known methods in the field of the art
can be su~.tab~,y used, e.g. random priming method, nick
translation method, DNA amplification by PCR,
labeling/tailing method, and in vivo transcxi.ption method.
The observation of the treated samples is performed by a
known method in the field of the art by using a dark-field
microscope, a phase differ~nt microscope, a refl.ecti.or~
contrast microscope, a fluorescence rnicroscop~, a digital
imaging microscope, or an electxoz~ microscope. Flow
cytometoryl can be also used.
3~ gene diagnosis (detection method) of
physiological phenomena during early pregnancy involved in
IF-10 is provided according to the present invention_ The
gene diagnosis may include the steps of (a) a step of
preparing a nucleic acid sample, (b) a step of amp7.ifying
the nucleic acid sample prepared in the step (a) by PGR,
nucleic acid amplification using l~tvtA polymerise, strand
displacement amplification, or the like to prepare nucleic
acid segments, which include mutations existing in the IP-
gene, tsnd (c) a step of detecting the presence of
mutation in the nucle~.c ac~.d segments in the step (b). The
methods for amplifying nucleic acid include gene
amplification process generally used, e.g_ pGl~ (including
RT,PCR), nucleic acid sequencE based aunplification method
(NASBA), transcx~.ptiore-mediated amplification method (TMA),
strand displacement amplification method (SDA). The target
of the amplification can be selected accoxd~.ng to the
objective. For example, a region containing a mutation,
which causes a functional decrease, of ZP-10 gene
nucleotide sequence of the present invention is one of the
objects for the amplification. Examples of such a region
is, but not limited tv, a region containing bases at a
predetermined position in the nucleotide sequence shown by
CA 02497497 2005-03-02
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sEQ ZD NO: 1. In the step (c) in above, any msthod for
detecting mutation known by those in the field of the art
can be used. For example, the detect~.on of mutations can
be performed by investigating the DNA fragment length by an
allele-specific PCR method (Ta,SpGR), but not limited to this.
The method far investigating the DNA fragment length can be
performed by usa,ng fluorescence DNA sequencer, or the like.
Hut it is not lim~.ted to this. Examples of the method xox
deteGtirig mutation used in tha.s step include a process for
detecting restxiction ft~agment length polymorph~.sm (RFLP).
xhe detection of mutation can be perfarmed by a known
method, e.g. hyb~cidization using an appropriate DNA
containing a mutation as a probe and a single-strand
conformation polymorphism (SSCP). The gene diagnosis of
IP-10 of the present invention can be performed by the gene
diagnasis according to the present invention to diagnose
expzession or polymorphism of z1~-10, which may be a
resistance or sensitivity factor relating to interaction
between conceptus and mother, e.g. sterility and
implantation d~.sorder. Fusthertnore, gene therapy method
fox decreasing a risk of the matter or the related
dysfunction based on the diagnostic results can be pro~trided.
zn the present specification, IP--10, its related
psotcins, fragments, and nucle~.c acids (including mRNA and
oligonucleotide) xncJ.uding DNR can be used for genomics and
pxoteomics technologies by alone or organically, and
further in combination with gene manipulation technology or
antibody manipulation technology (for example, an antisense
method, an antibody inc7.uding a monoclonal antibody, and a
transgenic animal) wh~.ch axe widely known in the fiEld of
the art. IP-10 variants can be used gor a function
analysis utilizing the dominant-negative effect, and can be
a~.ao applied RNA intarference (RNAi) technology using a
double-stranded RNA (dSRNA). Thus, gene polymorphisms,
ma~.n~.y single nucleotide polymorphisms (SNP), analysis, an
nucleic acid array ( ~,nc~,udj.ng a 17NR microarray; Mark Shena
CA 02497497 2005-03-02
.. 4.4. _
(Ed.), "Microarray Biochip Technology", Eaton Publish~.ng,
March ZOpO), gene expression analysis using a protein array,
gene function analysis, relating gene analysis, protein
interaction analysis, xe~.ating disorder analysis, and
therapeutic drug analysis are possible.
When RNAi technology is used, shoat interfering
RNA (siRNA) may be designed and introduced. When the siRNA
is designed, a 19-base strand, which starts with AA about
50 to x.00 bases downstream, preferably at least 73 bases
downstream of the start codon of target gene, is typically
selected. PrefexESbly, 5'- and 3'-UTR and in the vicinity
of start codon axe avoided, in some case, UTR-region can be
selected. The GC content of the selected region is about
30$ to 70~, preferably about 95~ tv 55~. A strand of two
bases (~fi,A) and 19 bases (i.e. 21 bases), for example
AA(N19) (N: arbitrary nucleotides), is screened for DNA
database such as NCBI (e.g. SLAST search) to identify the
specificity. A synthetic RNA having the selected strand
may be added dTdT (or UU) at the 3' side. Synthetic RNAs
of sense strand and the corxesporidirtg t~rstisense strand are
prepared to form dsRN~. The dsRNA is introduced into cells
or the lake. Examples of typical annea7.ing buffer solution
used in the dsRN,x~, preparation include, but not limited to,
30 mM HEPES-KOH (pH 7.A) containing 100 mM KOAc and 2 mM
MgOAc; and 10 mM Tris (pFi 7.5 to 8.0) containing 50 rnM Nr~Cl
and ~. mM EDTA. Typical cond~.t~.ons far the annealing are
heating at 95°C for 2 or 3 minutes and then graduaJ.J.y
cooling up to 37°C to 25°C in 45 minutes to 60 minutes, but
it i,s not limited to this. The prepared dsRNA can be
collected by extraction with phenol./chl.oxoform and
precipitation with ethanol. The introduction of siRNA into
cells of mammal or the like can be performed by a method
known in the field of the art or substantially the same
method thereof, such as a calcium phosphate method (e_g. F.
~. Graham et al., (1973) Virology, 52: 456), pEAE-dextran
rnathod (e.g. D. Warden et al., (1968) J. Gen. Virol., 3:
371), liposome method such as cationic lipid complex
CA 02497497 2005-03-02
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format~.on, eleatroporation (e. g. E. Neumann et al., (1982)
EM80 J, ~.: 841), microinacction, and biolytic particle
delivery method. The nucleic acid introduction is
techn~.cally improved to be efficiently performed by
transfection. The introduction can be performed by
commercially available kit according to the protocol
disclosed by manufacturers, e.g. znvitrogen Corporation and
QZACsEN Tnc., or distributors. Regarding siRNA technology,
E~.baahir et al., (2001) Nature, 4~.1: 494-498; Elbashir et
al., (2001) Genes Dev., 15: x.88-200: and Kazunari Tabirt~
(ed.), (2001) Jikken Igaku (Experi.mental Medicine), an
extra number, Protocol Series
(Functional Inhibition Experiments in Gene) Yodosha can be
referred to.
A DNA microarray accoxdirig to a nucleic acid
array technology may be used- Regarding the preparation ofi
miaroarrays, two methods are known, i.e. a method of
directly synthesizing oligonucleotides on a surface of a
solid carrier (on-chip method) and a method of immobilizing
oligonuclaotides ox polynucleotides that are synthesized in
advance on a surface of a solid carrier_ Both methods can
be appl~.ed to manufacturing microarrays used in the present
i.nventian. In the on~-chip method, a selective synthesis on
a predetermined shallow matrix region is possible by a
combination with the use of a protective group which is
selectively removed by light irradiatien, a
photolithography technology used in semiconductor
preparation, and a solid-phase synthesis technology
(masking technology: e.g. Fodor, S.P.A., (1991) Science
251: 767). OJhen oliganucleotides or polynucleotides
prepared in advance are immobilized on a surface of a solid
carrier, a functional group is introduced to the
oligonucleotides or polynucleotides and the resulting
oligonucleotides or polynucleotides are attached on a
treated surface of a solid-carrier to covalently bind (e_g.
~amture, J.B. et al., (1999) Nucl. Acids Res. 22: 2121-
2125; Guo, Z. et al., (1994) Nucl. Rcids Res. 22: 5456-
CA 02497497 2005-03-02
-46-
5465). In general, oligonucltotidms or po~.ynucleotides are
covalently bound to a treated surface of a solid carrier
via a spacer ox a cross-linker. A known method includes
the steps of arraying ma.aro pieces of polyacxylamidm gml on
a glass surface, then covalently binding synthetxo
oligonucleotides on the surface (Yershov, G. et al., (1996}
Proc. Natl. Aced. Sci. U.S.A. 94: 4913). Another method
includes the steps of arraying mi.cxoelectrodes on a s~.l~.con
mxcroarray, formi.nc~ a penetration layer composed of agarose
and stxeptavidin on the electrodes to provide a reaction
region, and immobilizing biotinized olxgonucleotides on the
reaction region by positively charging the region. In this
method, the fast and strict hybridization can be performed
by controlling the charge of the region (Sosnowski, R.G. et
a7.., (1997) Proc. Natl. Aced. Sci. U.S.A. 94: 1119-7.123).
In analysis using nucleic acid array, cDNA ~.xbrary is
uti~.xxed. DNAs, which are prepared by PCR technology, is
mounted on a substrate at a high~density by a spotting
device. Specimens arc ana~.yxad by hybridization with such
DNAs. In arraying, DNA are attachmd and arrayed at the
predetermined pos~.t~.on on a substrata of glass, silicon, or
plastic using a needle or pin, or using inkjet printing
technology. Signals that show the results of hybridization
an the nucleic acid array axe observed to obtain data. The
signals may be labels such as fluorescence (e. g. Cy3, CyS,
BODIPY, FITC, Alcxa Fluox dyes (trade name), and Texas red
(trade name}). Tho detection can be conducted using a
laser scannms. The resulting data can be processed with a
computer system equipped with a program of appropriate
algorithm. In DNA analysis using a typ~.cal microarray,
cDNA is synthesized using mRNA isolated from cells as a
template, and then amplixitd by PCR. zn this process,
labeled dNTP is incorporated to produce ~.abeled aDNA. The
labexed cDNA is contacted with a microarray. Comp7.ementary
DNA that hybridized to a capture probe (oligonucleotide or
polynucleotidm) on the microarray is detected. xhe
hybridization ~.s performed by dispensing a labeled cDNA
CA 02497497 2005-03-02
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solution to a 96-weld, ox 384-well plastic plate. The
amount ox the solution may range about 1 to 100 nL.
Preferably, the hybridization is performed at the ranges
from room temperature to 70°C for 6 to 20 hours. After the
hybridization, the plate is washed with a solution mixture
o~ a surfactant and a buffer to remove unreacted labeled
cDNA. Sodium dodecyl sulfate (SDS) is a preferable
suxfaat.ant. Examples of the buffer include a citric acid
buffer, a phosphoric acid buffer, a boric acid buffer, a
txis buffer, and a Goodrs buffer. The citric acid buffer
is preferable.
Tn protein array technology, a tagged recombinant
expression product protein may be used. Two-d~.mentianal
electrophoresis (2--DE), mass spectrometry (MS) for samples
i.ncJ.uding enzyme-digested fragments, stai.nxng technique,
isotope-labeling and analyais, and image processing
technique may be used. The MS includes electxospray
ionization (EST) and matrix--assisted J.aser
desaxption/ionization (MALDI) and MALD=,TOf MS, ~SZ-triple
quadruple MS, and ES=--iontxap MS mrxy be used. Therefore,
software, database, and the like that are obtained or used
as above concerning with function analysis of interaction
between conceptus and mother which correlates with TP-10
and antibody thereof can be included in the present
inventzan.
According to the present invention, antisense
oligonucleotide (nucleic acid) that can inhibit TP-7,0 gene
expression can be designed and synthesized based on a
nucleotide sequence i.n~oxmat7.on abput DNA encoding IP-10
which is cloned and detErmined. The antisense
oligonucleotide can be used in surveying ox regulating the
investigated IE-10 function. Such an oligonucleotide
(nucleic acid) can hybridize with mEtNA of IP-10 gene. The
mRNA can inhibit the function o~ mRNA ox oan regulate or
control IP-10 gene expression via interaction w~.th Z~-10
related mRNA. In some cases, the mRNA can regulate or
CA 02497497 2005-03-02
-4$-
control TP,1Q gene expression by controlling an expression-
control region. 0l.xganucleotides complementary to a
sequence selected from TP-10 xel~xted gone and
oligonucleotides capable of specifically hybridizing to TP,
related gene are useful for the regulation and control
of IP--10 gene in vivo and in vitro, and useful therapy or
diagnosis of diseases related thereof.
As in described above, the target IP-10 is
obtained by introducing IP-10 gene and recombinant DNA
molecule and expressing IP-10 according to the results of
the research by the inventors. A method using the results
fox researching and surveying function relating to
interaction between conceptus and mother is pfiovided. zn
connection with the found functions, recombinant or
transfectant substantially expressing IP-10 gene,
manufacturing method thereof, and their use are provided
according to the present invention.
An object of the present invention is td provide
a good method or a reagent kit thereof fox tha detection or
fractional measurement of IP-10, its gene, or producing
ceJ.ls thereof in a specimen for the research or survey of a
phenomenon and function relating to the interaction between
conceptus and mother using zP-10 gene, probes induced
thereof, or an ~,nh~.bitor of IP-10 if necessary. It is
understood that each reagent of the reagent kit that can
detect or fractionally measure zP-10, its gene, or
producing cells thereof are included in emboda.ment
according to the present invention. The object of the
present ~.nvention is to provide a method, reagent, or
diagnostic agent for monitoring factors or physiological
phenomena relating to metabolism of cel~.ular protein,
activating hormone precursor, activating conceptus
migration, promoting conceptus implantation on the uterine
wall, treating sterility, promoting pregnancy, introducing
conceptus to the uterus during implantation period,
CA 02497497 2005-03-02
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contxo~.ling interaction between canceptus and maternal.
system, and pxoaesses thereof by the detection ar
fractional measurement of ZP-10, its gene, and producing
cells thereof by using the above,mentianed method.
Therefore, it is understood that the various use
of the reagents in medical and physiological. fields, and
the uses of the reageents for research, zsnalysis, and
measurement of responses, symptoms, and diseases caused by
controlling xntoraction between conceptus and IP-10,
activating conceptus migration, pxomating conceptus
imp~.antation on the uterine wa7.7., treating sterility,
promoting pregnancy, introducing conceptus to the uterus
during impJ.antation period, and aontrollxng interaction
between conceptus and maternal system, and the use for
diagnosis, prevention, and therapy, these utx~.ization are
all included in embodiments according to the present
invention.
IP-10 and xt$ related substances may be factors
having important functions between canaeptus and mother
during early pregnancy. Eor a pregnancy (implantation) to
be establishcsd, mother must secrete IP-1.0 ~.~rx proper time
and in proper quantity as a response to a signal from
conceptus. The maternal. system establishes physiological
and immunological responding paths, and pregnancy
(implantation) progresses. when the response to the signal
is insufficient, pregnancy i.s z~ot established. Therefore,
the protein is useful for communication between canceptus
and mother. Namely, Drugs containing a member selected
from rP-10, variants, modifications, or derivat~,~sres may
cure sterility caused by the ~.nsuffxcient IP-10 activity.
polypaptides according to the present invent~.on are useful
as a pharmaceutical drug fox the treatment and/or
prevention of these diseases.
Fox example, when the biological activity in
cells is 3.nsutf~.cierit ar abnormal due to TP-10 decrease or
deficiency in the living body, the phenomena are improved
CA 02497497 2005-03-02
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by (A) administering a protein of the present invention,
(B) administering nucleic acid such as DNA of the present
invention to express the proteins of the present invention
in vivo, or (Cy transplanting cells transduced nucloic acid
such as DNA of the present invention so as to express the
protein in vivo. Namely, the pregnancy ratio may be
improved by inducing the IP-10 production or administering
IP-10.
According to the biologically functional analysis
of IP-10 of the present invention, a compound (agonist or
promoting agent) pxomotzng the function of IP-10 or its
salts can be used as useful medicine (including veterinary
med~.cxne) for curing and/or preventing a var~.ety of
diseases relating to biological activities descx~.bed above,
e.g. deficiency in pregnancy caused by a dysfunction of IP-
10. Contrary to this, a compound (antagonist or
suppressing agent) suppressing the function of IP-~.0 or its
salts can be used ds useful medicine to cure and/or prevent
an excessive IP-10 function and control of pregnancy.
By focusing the function og =P-10 according to
the present invention, IP--10 and its related polypept~.des
are useful for screening agon~.sts, antagonists, or salts
thereof to physiological phenomena relating to interaction
between conceptus and mother. The prevent invention
provides a screening method of such agonists, antagonists,
or salts thereof, which promote or inhibit the
physiological. or biological activities.
In the screening method, for exampJ.e, (i)
contacting conceptus with protein, its partial peptide, or
a salt thereof (transformant expressing the pxotein may be
included; the same shall apply thereinafter) according to
the present invention, (ii) contacting conceptus and a test
sample with the protein, its partial peptide, or a sa7.t
thereof, and results in (i) arid (ii) are compared.
Specifically, a biological activity (e. g. act~.vi,ty relating
to xritoraction between conceptus and uterine membrane) is
CA 02497497 2005-03-02
-~1-
measured in the screening method, and compared. A
substrate instead of conceptus can be used. The substrate
can be used as it ~.s, preferably, the substrate labeled
wi'~h a fluorescence such as tluoresceine, an enzyme, or an
radioisotope ~.s used.
Examples of the test sample inaZude proteins,
peptides, non-peptidic compounds, synthetic compounds,
fermentation products, plant extracts, tissue extracts of
animal, cell extracts. Examples of the test sample include
anti-IP-10 antibodies, anti-CXCR3 antibodies, anti-TFN-t
antibodies, inhibitors of binding between CXCR3 and TP-10,
compounds for inhibiting conceptus implantation, ~xnd
compounds for promoting conceptus implantation, and
especially synthetic compounds thereof. These compounds
may be novel ox known. The screening can be performed
according to an ordinary method for measuring a binding
activity. Methods known in the field o~ the art can be
used. Naunely, methods are performed according to the
protocols using various labels, buffer solutions, and other
appropriate reagents. The peptides that are used in the
measurements may be treated with an activating agent, or
rnay be activated in advance from precursors or potential
types. The measurement is usually performed in a buffer
solution thnt does not affect the reaction. For example,
tris-HCl buffers and phosphate buffers, at pIi about 4 to 10
(preferably pti about 6 to 8) can be used. Actual screening
systems may be constructed with relation to TP-10,
polypept~.des or peptides which have substantially the same
activities as those of IP--10 of the present invention, with
general technique known by those ~.n the arts as well as
ordinary conditions and manipulations of individual systems.
Details of these general technological means can be
referred to review articles and monographs (fox example,
Methods in Enzymology, Academic Press, U.S.A.).
CA 02497497 2005-03-02
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Compounds or the~.x salts which are obtained by
the screening method or using the screening kit according
to the present invention are selected ~xom the abvvc-
ment~.oned test compounds, e.g. peptides, proteins, non-
peptidic compounds, synthetic compounds, fcrmontation
products, cell-extract solutions, plant-extract so~,utxons,
and animal-tissue-extract solutions. These compounds
promotes or inhibit functions of proteins of the present
invention. Examp7.cs of pharmacologically accEptable salts
of the compounds include salts with inorganic base, sa~.ts
with organic base, salts with inorganic acid, salts with
organic acid, and salts with basic or acidic amino acid.
Preferable examples of the salts with inorganic base
includ~ salts with alkali metal such as sodium and
potassium, salts with alkaline--earth metal such as calcium
and magnesium, salts with aluminium, and salts with
ammonium. Preferable examples of the salts with organic
base include salts with trimethylamine, triethylamine,
pyxi,dXnc, picoline, 2,6-lutidinc, cthanolaunine,
diethanolamine, triethanolamine, cyclohexylamine,
dicyclohexylamine, and N,N~-dibenzylethylenediamine.
Preferable examples of the salts with inorganic acid
a.nc7.ude salts with hydrochloric acid, hydrobronic acid,
sulfuric acid, and phosphoric acid. Preferable example of
the salts with organic acid include salts with formic acid,
a~ceti.c acid, propionic acid, fumaric acid, oxalic acid,
tartaric acid, maleic acid, citric acid, succinic acid,
malic acid, methanesulfon~.c acid, benzenesulfonic acid, and
benzoa.c acid. pxe,fexab~.e examp3.es o,f the sa~.t.s w~,th bas~.c
amino acid include salts with arginine, lysine, and
ornithine. Prcfcrable examples of the xalts with acidic
ama.no ac~.d ~.nGJ.ude salts wa.th aspaxtic acid and g7.utaxnic
acid.
rn the present s~pecificatxon, the term "antibody,
may be used in a broad sense. Thus, it includes individual
monoclonal antibodies to tha deai.rad ~p--7.0 polypepti.des and
CA 02497497 2005-03-02
-53-
related peptide fragments, and antibody compos~.tions having
specificity for various epitopes. It also includes
univalent antibodies and po~,yva7,er~t antibodies as well as
po,lyclonal antibodies and monoclonal antibodies_
Furthermore, it includes the na~t~.ve ( intact ) mo~.ecu7.es, and
Fragments and derivatives thereof, i.e. such fragments as
f(abt)2, Fab~, and dab and further, chimera antibodies or
hybrid antibodies having at least t."to antigen or epitope
binding sites, or bispecific recombinant antibodies ouch as
quadromes and txiomas, interspecific hybrid antibodies,
anti-idiotype antibodies and, further, chemically modified
or processed ones which can be considered to be dexxvatXves
of these, ant~.bodi,es obta~,ned by applying a per se known
ae~.~. fusion or hybridoma technology or antibody engineering
technology ox ut~.lxxxng a~ synthotic or semisynthetic
teChn,ology, antibodies prepared by applying conventional
technology known as antibody-preparing method by using a
recombinant DNl~ techz~oJ.ogy, snd antibodies having
neutralizing characteristic or binding chaxactexxsti.c
against the target a~ntigøn substance or target epitope as
described herein. A particularly preferred antibody
according to the present invention can specifically
recognize the native IP-lb polypeptide.
xhe monoc~.onal. antibody produced against the
antigenic substance can be produced by any of the methods
capable of causing the production of antibody molecules in
a series of cell lines under cultivation. The modifier
~~monoclona3." ~.ndicatps the chaxactexiatic of an antibody
that belongs to a substantially homogeneous antibody
popu~.at~.on. It is riot to be construed that the antibody
should be produced by a certain specific method.
individual monoclonal antibodies each includes a population
of the same antibod~.es except that a slight amount of a
possible mutant being spontaneously formed may be present
therein. Monoc~.onal antibodies each have high specif~.city
and axe directed to one single antigenic site. As compared
with ordinary (polyclonal) antibody prepz~rations typically
CA 02497497 2005-03-02
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containing various ant~.body speaios directed to different
antigez~xc detormina~nts (epitopes), each monoclonal antibody
is directed to one singla antigenic determinant on the
antigen. Tn addition to their specificity, monoclonal
antibodies are synthesized by hybridoma culture and are
superior in that they are not or only a little contaminated
with other immunoglobulins. the monoclonal antibodies
include hybrid antibodies and recomb~.nant antibodies. So
long as they show the desired biological activities, a
constant region domain may be substituted for variable
region domain thereof (for example, humanized antibody), or
heavy chain may be substl~tuted for a light chain thereof, a
chain derived from a certain species may be rep~.aced with a
chain derived from another species, yr they~may be fused
with a heterogeneous protein, irrespective of their origin
or immunoglobulin class or subclass (e.g. U.S. Patent No.
4,816,567: Monoclonal Antibody Production Techniques and
Applicz~tions, pp.79-97, Marcel Dekker, Inc., Naw York,
1987).
As exemplary method xox producing the monoclonal
antibody, there may be mentioned the hybri.doma method (G.
Kohler and C. Milstein, (7.975) z~ature, 256: 495-497); human
B cell hybridorna method (Kozbor et al., 0.984) Immunology
Today, 4: 72-79J Kozbox, (19$4) fir. xmmunvl., 133: 3001);
8xo~leur et al., (1987) Monoclonal Antibody Production
Techniques and Applications, pp.51,63, Marvel Dekker, rnc.,
New York; trioma method; EBV-hybridoma method (Cole et al.,
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp.77-96)(methods For producing human
monocJ.onal antibodies): U.s. Patent No. 4,946,778
(technology for producing single-chain antibodies) and,
further, the following references may be cittd for
antibodies: S. Biocca et al., (199b) EMBO J, 9: 1~1-108;
R.E. Bird et al., (1988) Science, 242: 423-426; M.A. Boss
et al., (1984) Nuc~.. Acids Res., ~,2: 3797.-3$06j J. Sukovsky
et al., (1987} Hybridoma, 6: 219-228; M. DAINO et al.,
CA 02497497 2005-03-02
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(1987) Anal. Biochem., 166: 223--229; J.S. Huston et al.,
(1988) Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883; P.T.
Jones et al., (1986) Nature, 321: 522-525: J.'T. Langone et
al. (ed.), "Methods ~.n En2ymology", Vol. 121
(Immunochemical Techniques, Part T: Hybridoma Technology
and Monoclonal Antibodies), Academic Press, New York
(1986) 5. Morrison et al., (1984) Proc. Natl. Acad. Sci.
U.S.A., $1: 6851-6855: V.T. Oi et al., (1986) B~.oxeGhnitjuess,
4C: 21.4221; z. RieGhmann et al., (1988) Nature, 332: 323-
327: A. Tramontano et al., (7.986) Prvc. Natl. AGad. Sci.
U.S.A., 83: 6736-6740: C. Wood et al., (1985) Nature, 37.4:
946-949: Natuxe, 314: 452-454: and references cited therein
(the description therein are incorporated herein by
reference).
The monoc7.onal antibody according to the present
irsventivn particularly includes "chimera" antibod~.es
(immunoglobulins), in wh~,ch paxt of the heavy chain and/or
light chain has a sequence identical or homo~.ogous to the
corresponding sequence of an antibody dcrivod from a
specigi.a species or belonging to a specific antibody class
or subclass while the rema~.ning poxt~.ons are identical or
homoJ.ogous to the corresponding sequences of an antibody
derived from another species or belonging to anothex
antibody class or subclass, so long as they show the
desired biological activities (U. S. Patent No. 4,87.6, 567;
Morrison et al., (1984) Proc. Natl. Acad. Sci. U.S.A., 81,
pp.6851-6855).
Ant~,body may be fxac~ments, e-g- Fab, Fab', and
f(ab')z, obtained by treatment of the antibodies with an
enzyme such as trypsin, papain, or pepsin, a.f necessary
followed by reduction.
The antibodies can be used in any known assay
method, ~r.g. oom~etitive binding assay, direct and indirect
sandwich assay, and immunoprecipitation [Zola, Monoclonal
Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,
Inc., 1987)].
CA 02497497 2005-03-02
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For conjuqatxng the antibodias to a detectable
atomic group, any m~thod known in the field of the a,rt can
be used. for axample, David et z~l., (Z974) Biochemistry,
13: 1024-1021: Pain et al, (1981) J. TmmunoJ.. Meth., 40:
219-231; and "Methods in Enzymology", Vol. 184, pp.138-163
(1990) are mentioned. Aa the antibodies to be labeled, IgG
fraction and the spee~.fic bind:tng portion ~al~' obtained by
reduction-following pepszn digest can be used. In these
cases, examples of ~Che label include enzymes (e. g.
peraxidase, alkaline phosphatase, or S~-D--galactosidase),
chemi.ca~, substances, fluorescent substances, and
radioisotopes.
The detection and assay according to the present
invention can be performed by i.mmunostairiing, e.g. tissue
or cell sta~.n~.ng and i.mmuno--electron microscopy: and
immunoassay, e.g. compctitivc immunoassay and
noncompetitive immunoassay. Radioimmunoassay (RxA),
fluorescent immunoassay (FrA), luminescent immunoassay
(LIA), enzyme immunoassay (ETA), enzyme-linked
immunosorbent assay (EL7Csl~) , and the ~.ike can k~a used.
.il~ssaying may be performed with or without B-F separation.
RIA, EIA, fxA., arid x.xA axe pxaferable, and sandwich rassay
is preferably used. In the sandwich assay, simultaneous
sandwich assay, forward sandwich assay, and reversed
sandwich assay may be included.
Examples of the label include enzymes, enzyme
substrates, enzyme inh:Lb3.tors, prosthetic groups, co~nzymes,
enzyme precursors, apoenzymes, fluor~scent, substances,
chromophores, ehemiluminescent compounds, luminescent
substances, chromogens, magnetj.c substances, metal,
particles such as colloidal gold, and radioisotopes.
Examples of the enzyme include oxyreductase such as
dehydrogenases, reductases, oxidases; transferases
cataJ.yzzng the transfer o.f arn3.no, carboxyl, methyl, acyl,
or phosphoryl group: hydrolases hydrolyzing the es'~es,
glycoside, ether, or peptide bond: lyases: isomerases; and
ligases. A plurality of enzymes may be utilized in
CA 02497497 2005-03-02
combination fox detect~.on purposes. Fox examp7.e, enzymatic
cycling can by utilized.
A biotin label and enzyme-labeled avidin
(streptavidin) may be substituted for the enzyme label. A
pluxaJ.ity of different labels can be used. In such case, a
plurality of measurements can be performed continuously or
discontinuously, and s~.muJ.taneougl.y ox separate~.y.
xhe label~.ng, can be pexfoxrned ut7.7.~.axng the
reaction between a thiol group and a maleimide group, the
reaction between a pyridyl disulfide group and a thiol
group, or the like that is known or obvious to those
skilled in the art, or modifications thereof.
Regarding the assaying xP-7.0 ox the like
according to the present invention, tissue samples are
advantageously assayed by protein assa:yi.ng systems such as
immunostaining and immuno-electron microscopy and express~d
gene assaying systems such as in situ hybridization; tissue
extracts are assayed by protein assaying systems such as
ETA, RTA, FTA, LTA, and Western blotting and expressed gene
assaying systems such as Northern blotting, dot blot, RNase
protection assay, RT-PCR, Real-Time PCR, and competitive
PCR, and blood, body fluids are a~ssa~yed by protein assaying
systems such as EIA, RIA, FIA, LTA, and Western blotting.
For example, in ETA systems using a competitive method, the
anti,-zP-7.0 antibody ~,s used as an immobilized antibody and
a labeled antigen and an unlabeled antigen (ZP-10 or a
fragment peptide thereof may b~ m~ntionsd as the antigen)
are used. In the case of a non-competitive method such as
sandwich method, an immobilized anti-IP-10 antibody and
labeled anti-rP-10 antibody may be used. A labeled anti-TP-
may be used, or a labeled antibody to the anti-IP-10
antibody may be used. Regarding the sensitivity-enhancing
method, there may be mentioned tha uti7.i.zatxon of, in the
combination with a non-enzyme--labeled primary antibody, a
macxomo~.ecuJ.ax polymer and an enzyme and the primary
antibody [application of Envision reagent: Enhanced polymer
one-step staining (EPOS)]; and, in the combination with a
CA 02497497 2005-03-02
non-enzyme-labeled secondary ant~.body, the combination o~
an enzyme and an anti-enzyme antibody as in the peroxidase-
ant~.pexoxidase technique (PAP), the combination of a
biotin-labeled secondary antibody a,nd a biotin-labeled
enzyme-avidin complex as ~.n the avidiri-biotin oomplex
method (ABC), the combination of a biotin-labeled secondary
antibody and a biotin-labeled enzyme-streptavidin complex
as in the streptavidin~biotinylated perox~.dase complex
method (SABC), the labeled streptavidin-biotin method
(LSAB), the combination of a biotin-labeled tyxamide and an
enzyme-labeled streptavidin on the SABC as in the catalyzed
signal. ampl~.f~.cation method (CSA) , and the use of a
maaromolecular polymer labeled with a secondary antibody
and an enzyme.
When these vaxi.ous analyses and quantitative
assays a.z~cludin~ immunoassays are applied to the measuring
methods according to the present invention, particular
conditionac and manipul~xtions are not necesasrily
established. According to typical conditions and
manipulations in each method as well as typzcal tachniaal
arrangements by those skilled in the art, assay systems may
be estab~.~,shed in relation with the target substances or
substances having substantially equivalent activities
thereof.
For the details of these general technological
means, xev,iew axt~.c~.es and monagraphs may be xeferred to
[e. g. Hiroshi Irie (ed.), "Radioimmunoassay", Kodansha,
published in 1974: i3iroshi Irie (ed.), "Radioimmunoassay,
supplement", Kodansha, published in 1979; Eiji Ishikawa et
al. (ed.), "Koso Men'eki Sokuteiho (Enzyme immunoassay)"
Igaku Shoin, published in 2978; Eiji Ishikawa et al. (ed.),
"Koso Men'ekz Sokute~,ho (Enzyme immunoassay)" (second
edition), Igaku Shoin, published in 1982: "Koso Men'eki
sokuteiho (Enzyme immunoassay)" (third edition), Igaku
Shoin, published in 1987; H. v. vunakis et al. (ed.),
"Methods in Enzymol.ogy", Vol. 70 ( Tmmunochemical Techniques,
CA 02497497 2005-03-02
- 59 -
Part A), Academic Press, New York (1980) J. J. Langone et
al. (ed.), "Methods in Enzymology", Vol. 73 (Tmmunochemical
Techniques, Paxt B), Academic Press, New York (1981): J. J.
Langone et al. (ed.), "Methods in Enzymology", Vol. ?4
(Immunochemical T~chniques, Part C), Academic Press, New
York (1981); J. J. Langone et al. (ed.), "Methods in
Erizymology", Vol. 84 (Immunochemical Techniques, Part D:
Selected Immunoassays), Academic Press, New York (1982): J.
J. Langone et al. (ed.), "Methods in Enzymology", Vol. 92
(Immunochemical Techniques, Part E: Monoclonal Antibodies
and General immunoassay Methods), Academic Press, Nevi York
(1983) s J. J. Langorle et aJ.. (ed. ) , "Methods in Enzymdlogy",
Vol. 12~. (~mmunochemi.cal Techniques, Part ~: Hybridoma
Technology and Monoclonal Antibodies), Academic Press, New
York 0.986): J. J. Langone et al. (ed.), "Methods in
Enzymology", VoJ.. 178 (Ant~,bod~,es, Antigens, and Mol.ecul.ax
Mimicry), Academic Press, New Yark (1989); M. Wilchek et al.
(ed.), "Methods in Enzymology", Vol. 184 (Avidin--Biotin
Technology), Academic Press, New York (1990); J. J. Langone
et al. (ed.), "Methods in Enzymology", Vol. 203 (Molecular
Design arid Modeling: Concepts and Applications, Part 8:
Anibodies and Antigens, Nucleic Acids, Polysaccharides, and
Dxugs), Academ~.c Pxess, New York (1991), and references
cited therein (the description therein are incorporated
herein by reference ) .
When active ingredients of the present invention.
are used as medicine [for example, (a) zp-10 polypeptide,
its partial peptides ox ~.ts salts, and related peptides
thereof, (b) nucleic acids, e.g. DNA encoding the zP-10 or
zP-10 polypeptides, (c) antibodies according to the present
i:lvent~.otx, paxt~.a~, fragments thereof (including monoclonal
antibodies), or derivatives thexeof, (d) compounds or their
salts promoting and/or activating biological activities
such as interaction between conceptus and mother (e. g.
implantation) by IP-10, and denaturation, excessive
production, and decomposition of the related tissues and
CA 02497497 2005-03-02
-~?~-
proteins; compounds or their salts controlling the
production of IP-10: and compounds or their salts
suppressing and/or preventing biological activities such as
interaction between conceptus and mother (e. g.
implantation) by TP-10, and denaturation, excessive
production, and decomposition of the related tissues and
proteins, (e) antisens~ oligonucl~otides to th~ nucleic
acids of DNR according to the present invention, (f) active
substances found by utilizing the present invention], the
ingredients can be administered alone or combination with
pharmacologically acceptable adjuvarsts as med~.cal.
components or medical preparations ~or, ~ox example,
promoting interaction between conceptus and mother.
Preferably, they are administered in the forms of dosage
preparations suitable for the use in oral administration,
local admxnistxatxon, ox parenteral administration.
Administration forms (including inhalation, rectal
administration) may be determined according on the
objective.
The active ingredients of the present invention
may be used in combination with substances known in the
field of the art, such as hormone effectors, vitamins,
cytokinos, interferons, other physxoJ.og~.cal7.y active
substances and/or immunomodulator, which are not limit~d so
long as they have a useful function.
Examples of the p~arenteral administration form
include local, transcutaneous, intravenous, intramuscular,
hypodermal, intradermal, and infra-abdominal adm~.ni.strat~.on.
Direct administration to affected area is possible, and it
~.s su~.tab~.e ~.n some cases. Preferably, they are
administered to mammals including human orally or
parenterally (e. g. intracellular, intarrtal, i.ntxavenous,
intxamusculax, hypodermal, intradermal, intro-abdominal,
intro--thoracic administration, spinal cavity, instillation,
intestinal infusion, through the xecturn, ear drop, eye drop,
nose drop, and apply to teeth, skin and mucous membrane).
CA 02497497 2005-03-02
- Gl -
Examples of the preparation form include liquid, suspension,
semi-sol~.d, powder, shaped, and exudat~.ve foams, e.g.
tablets, coated tablets, sugar-coated tablets, balls,
troches, hard-capsules, soft-capsules, micxocapsules,
embedd~.ng, powders, dispexsi,ble powders, granules, fine-
powders, injections, liquid, elixirs, emulsions, affusions,
syrups, liquid, emulsions, suspension, liniment, lotion,
aerosol, spray, inhalation, atamiza, ointment, hard
ointment, plaster, paste, cataplasrn, cream, oleum,
suppository (e.g. rectum suppository), tincture, liquid for
skin, eye drops, nose drops, ear drops, swab, infusion,
powder for inject3.on, lyophilized preparations, and gel
preparations.
Drug components aro propared acdording to grnerxsl
methods. Optionally, physioJ.og~.cal7.y acceptable carriers,
pharmaceutically acceptable carriers, adjuvants, excipients,
Forming agents, d~.luents, flavoring agents, essence,
sweeteners, vehicles, antiseptics, stabilizers, binders, pH
regulators, buffers, surfactants, bases, solvents, fillers,
extenders, solubili,zing supplements, solubilizexs,
isotonizing agents, emulisi~iers, suspending agents,
dispersing agents, thickeners, gelling agents, st~.ffenxnc~
agents, absorbents, adhesives, flexing agents, plasticizers,
collapsing agents, em~.tti.ng agents, preservatives, anti-
oxidants, shading agents, lubricants, mitigatives,
antistatic agents, analges~.c agents are used alone or
combination. These additives are mixed with proteins yr
the like of the present invention so that unit doses which
are x~qu~.xed in typical drugs are prepared.
Examples of the pa~renteral drugs include
xrijeat~.ons such as abacterial solutions or suspensions of
active ingredients and water or other pharmaceutically
acceptable solvents. preferable examples of carrier of the
injections generally include water, saline, dextrose
solutions, other sugar solutions, ethanol, and glycols, e_g.
propyleneglycol and polyethyleneglycvl. An iri7ection
solution, suspension, or emulsion is prepared by methods
CA 02497497 2005-03-02
-62-
using n carrier such ns distilled water, Ringer s solution,
or sal.i.ne, a d~.spersing agent ar maisturizing agent, and a
suspending agent, which are known in the field o~ the a,xt.
Since polyethyleneglycol (PEG) has very low
toxicity in mammals, the canjugatian o~ BEG is particularly
use~u1. The conjugation of PEG may effectively reduce the
immunogenicity and antigenicity o~ hetexologaus compounds.
The compound may be del.ivexed in a microcapsulat,ion dev~.ce.
Polymers such as PEG can be readily attached to a-amino
groups of the amino-terminal rxmino acids, ~-amino groups of
lysine side chains, carboxyl groups of aspartyl and
glutamyl side chains, a-cazbvxyl gxoups o~ ca~xboxy-tex-rttirisl
amino acids, or to activated derivatives of glycosyl chains
attached serirte or threonirle xesidues.
Numerous activated forms of PEG for direct
xeaction with proteins axe known. Examples o~ useful PEG
reagents for reaction with amino acid groups of proteins
include act~.ve esters of carboxylic acid or carbonate
derivatives, particularly those in which the leaving groups
axe N-hydxaxyauca~.n~,m7.de, p--ni.trophenol, imidazol,e, or 1-
hydsoxy-2-nitrobenzene-4-sulfonate. likewise, pEG reagents
containing amino hydrazine ox hydxazide gxoups axe useful.
to the reaction with aldehydes generated by periadate
oxi.dat~.on o~ proteins .
When nucleic acid such as DNA of the present
invention is used for the treatment and/or pxevent~.ve
medicine, the nucleic acid can be used a~l.one or combination
with the above-mentioned appropriate nectar used in gene
xecomb~.nation technology, for example virus-derived vectors,
a . g , retrovirus-derived ~crectars . xhe nuc7.ezc ac~.d such as
DNA of the present invention can be administered by well-
known methods as it is or as a medicine component or
medicine preparation with appzopx~.ate adjuvants or
phys~.ol.ogi.cally acceptable carriers so as to promote the
intake into cells. Methods known as genetic thexapy are
applicable.
CA 02497497 2005-03-02
- 63 -
Active ~.ngredients according to the present
invention can be administered in various doses. Doses and
frequenaa.es axe determined depending on sex, age, weight,
general conditions of a subject, meal, administration time,
administration method, excretion rate, combination with
other medicine, degrees of symptoms of the subject at the
treatment, and other factexs.
Regarding the manufacturing of the medicine, the
Japanese pharmacopoeia commission (ed.), "Manual of
Japanese Phaxxnacapvoia 14th edition", published on June ~7,
2001, H~.xokawa Shoten; and zchibangase Naoshi, et sl.,
(end.) "Iyakuhin No Kaihatsu (Development of
Pharmaceuticals) Val. 12: seizai sozai [II] (Pharmaceutical
Necessities [zI])", pub~.i.shed ari October 28, 1990, Hirokawa
shoten axe referred, and additives and preparation methods
optionally selected from the descriptions therein can be
applied.
The active ingredients according to the present
invention typically have biological act~.vities to promote
or activate interaction between conceptus and mother.
Preferably, such ~.ngxedxents show advantageous functions.
Examples of the native ingredients according to the present
invention include (a) IP-10, its variant polypeptides,, or
paxtzal peptides ar salts thereof, (b) nucleic acids, e.g.
DNA encoding the IP-10 or IP--10 variant polypeptides, (c)
antibodies according to the present in~r'ention, partial
fragments thereog (including monoclonal antibodies), ox
derivatives thereof, (d) compounds or salts thereof having
at least one advantageous fiunction such as act~.vatxng
corsceptus migration, promoting conceptus implantation on
the uterine wall, treating sterility, promoting pregnancy,
activating migration of conceptus to uterus during an
implantation period, and controlling interaction between
conceptus and mother.
The active ingredients according to the present
invent~.on axe expected as agents using biological
CA 02497497 2005-03-02
-64-
activities, e.g. activating conceptus migration, promoting
conceptus a.mp~.ar~tativn on the uterine wall, treating
sterility, promoting pregnancy, introducing conc~ptus to
the uterus during an implantation period, and controlling
interaction between conceptus and mother, and are expected
to be useful for a preventive or treating method.
Zn the present invention, predetermined nucleic
acids are iaoxated and sequenced by "gene recombination
technology", recombinants are prepared, arid predetermined
peptides are obtained. A gene recombination technology
known in the field o~ the art may be used in the present
spec3,fication. For example, the methods described in: ,T.
Sambrook, E. f. Fxitsch b~ T. Maniatis, "Molecular Cloning:
A Laboratory Manual", (2nd edition, 1989 & 3rd edition,
2002), Gold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York: D. M. Glover et al. ed., "DNA Clotting",
2nd ed., Vo~.. 7, to 4, (The L~ractical Approach Series), IRL
Press, Oxford Univars~.ty Press (1993); "Methods in
Enzymology" series, Academic Press, New York, for example,
R. Wu ed., ibid., Vol. 68 (Recombinant DNA), (1980}; R. Wu
et al. ed., ibid_, Vol. 100 (Recombinant DNA, Part S) & 101
(Recombinant DNA, Part C), (1983); R. Wu et al. ed., ibid.,
Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA,
Part E) & 155 (Recomb~.nant DNA, Part F), (1987); J. H.
Mi~.ler ed. , ibid . , Vo7. . 209, ( 1991 ) : R. Ortu ed . , ibid . , Vol .
216 (Recombinant DNA, Part G), (7.992): R. Wu ed., ibid.,
Vol. 217 (Recombinant DNA, Part H) & Z18 (Recombinant DNA,
Part I}, (1993); J. L. Campbe~.l, ed., ibid., Vpl. 262 (DNA
Replication), (1993); P. M. Gonn ed., ibid., Vol. 302
(G~tetn fluorescent Protein), (1999); S. Weissman ed., ibid.,
Vol. 303 (cDNA Preparation and Characterization), (1999): J.
C. Glorioso et al. ed., ibid., Vol. 306 (Expression of
Recombinant Genes in Eukaxyotzc Systems), (1999): M. lan
Phi111ps ed., ibid., Vol. 313 (Antisense Technology, Part
A: General Methods, Methods of Delivery and RNA Stuct~t~rs) &
319 (Antisense Technology, Part B. Applications), {1999); J.
xhoxnex et al. ed., ibid., Vol. 326 (Applications of
CA 02497497 2005-03-02
Chimeric Genes and Hybrid Proteins, Part 1~_ Gene Expression
and Protein Purification), 327 (Applications of Chimeric
Genes and Hybrid Proteins, Part 8: Cell Biology and
Physiology) & 328 (Applicat,ions of Ch~.meric Genes and
Hybrid Proteins, Part C: Protein-Protein xntexactxoris and
Genornics), (2000), and methods described in references
cited therein and substantially the same methods or
medxficativns thereof are mentioned (the description
therein are incorporated herein by reference). The
preparation of the reagents used in the diagnosis methods
according to the present invex~txon are performed by methods
described in, for example, A. Gennaxo ed., "Remington's
Pharmaceutical Sciences", 18th Edition, Mack Publishing Co.,
Easton, PA, 1990, and by molecular biological techrso~,oc~y
described in, for example, Ausubel, F. M. ct al., "Current
Protocols in Molecular Biology", John Wiley & Sons, New
York, N.Y, 1995, and methods or technology described in
references cited therein and substantially the same methods
or medifications thereof are mentioned (the description
therein are incorporated herein by reference).
In the specification and figures, terms according
to xt~pfi.C-ZtJE Commission on Biochemical Nomenclaturm or the
usual practice in the field of the art are used.
Examples
Details of the prevent invention are described by
the following examples but such examples are provided only
xor ~.llustrative purposes, and fox referential emb4a~.ments
of the present invention. These examples have been
described herein for the purpose of illustrating specific
exnbod~.menta of the present invention but should ~.n no way
be construed as limiting and restricting the scope of the
invention disclosed herein. It should be understood in the
present invention that various embodiments can be made or
CA 02497497 2005-03-02
.-6b-.
executed within the spxxit, scope arid cvnGept disclosed
herein.
All the examples were cs~xried out or can be
carried out, unless otherwise disclosed herein specifically,
by standard techniques which are well known and
conventional, to those sk~.xXed ~.~, the field of the art.
The concrete manipulations and treatment
conditions in the following Examples are performed, unless
indicated particularly, acco7cding to methods described in J.
Sambrook, E. F. Fritsch ~ T. Maniatis, "Molecular Cloning:
A Laboratory Manual.", (2nd edition, 1989 & 3rd edition,
2001), Cold Spring Harbor Laboratory press, Cold Spring
Harbor, New Xork and 17. M. Glover et al. ed., "DNA Cloning",
2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL
Press, Oxford University Press (1995) for DNA cloning: and
H. A. Erlich ed., PCR Technology, Stockton Press, 7.989 t D.
M. Glover et al. ed., "DNA Cloning", 2nd ed., Vol. 1, (The
Practical Approach Smri~s), zRL Press, Oxford University
Press 0.995) and M. A. rnnis et al. ed., "PCEt Protocols",
Academic Press, New York (1990) for PCR method.
Commercially available reagents and kits are used according
to the attached protocols using the attached substances.
Exami~le 1
[Animal and tissue preparation]
Whitefaaed crossbred sates were maintained at the
US Meat Animal Research centar (Clay Center, NE, U.s.A.).
The protocol for sheep experimentation has been reviewed
and approved by the animal cares committees at the USDA.
Animal care and estrous synchxon~.zat~.on
procedures were performed as in Ima~kawa K. et al., Endocr.
J., 45: 441--450 (1998). Uteri from cyclic ewes of on Day
15 ( n = 9 ) and pregnant ewes on Days 7.4 ( n = 3 ) , 17 ( n = 9 ) ,
20 (n = 3), 25 (n = 3), and 30 (n =3) were removed
immediately after slaughter. Whole uteri from Day 15
CA 02497497 2005-03-02
..67-
cyclic and Day 17 pregnant ewes (n = 3 each) were
immobilized as in Imakawa K. et al., Endocx. ,7~., 45: 441-
450 (1998), and then frozen immediately for subsequent in
situ hybxid~.zat~.on studies. Fndometrial and conceptus
(embryos and embryonic membranes) tissues collected from
the remaining pregnant ewes (n = 3 for each day examined)
were frozen and stored at --70°C. These frozen tissues were
used far RNA extraction. At the University of Tokyo farm,
whole bl4ad was Collected from three cyclic ewes.
Eeripheral blood mononuclear cells (PBMCs) were obtained
from the whole blood. These samples were used for IFN dose
responss~ and chemotaxis assays. Whole uteri were obtained
from three additional cyclic ewes, and endometrial txplants
were cultured to examine the stimulatory effect of IFNs on
IP-10 production. The use of sheep has been approved by
the animal care committee at the Unxvexsi.ty of Tokyo.
Animal care and estrous synchronization were performed
accordingly as in Aida H. et al., J Peprod. Dev., 45: 249-
257 (1999)
Czn vitro culture?
Primary monocytes and lymphocytes were ~.sozated
from Day 15 cyclic ewes using a method (Domeneah A. et al.,
(2000) J Gen. Virol., 81: 109118) with minor modification.
L~SMCs were separated from EDTA-treated blood (EO mL) by
density gradient centrifugation (800 % g, 20°C, 30 min,
4ptiPrepTM, Nycomed, Rvakilde, Denmark) and were auspendr~d
in RPMI 1690 medium supplemented with 10$ fetal calf serum
(FCS), 90 units/mL of penicillin, ~O~c/mL of streptomycin,
and anti-pleuropneumonia-like organisms (PPLO: Invitrogen
Corp., Cax~.sbad, CIa,, U.S.A.). QBMCs (3 x 107 cells/mh) were
plated ~,n 6-well coaster plates (~ mL/well), which were
incubated at 37°C in a 5~ C42-95~ air atmosphere fox 2
hours. The adi~exent cells (monocytes) were separated from
nonadherent cells (lymphocytes) and then cultured in the
fresh RPMZ 1640 medium with same supplements as above. To
determine effective dorms of IfNs on YP ~10 expression,
CA 02497497 2005-03-02
_(~8_
monocytes were treated with 102, 103, or 104 TU/mL
recombinant human TFN- a (rhIFN- a, Sigma, St. Zouis,
Missouri, U.S.A.), recombinant human ZFN- y (rhIFN- y,
Upstate biotechnology, Lake Placid, I~ew York, U.S.A.), or
recombinant bovine IFN--z ~(rbIFN-s, Katakura Industries
Co.,). After 20 hours at 37°C in a 5~ C02-95'k atmosphere,
culture media were harvested and stored at ~-70°C, and cells
were i.mmed~.ately processed for total RNA extraction. Ovine
uterine epithelial and stroma cells (Johnson G.A. et al.,
(1999} BxoX. Reprod., 61: 1324-1330} were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
40 units/rnL of penxaxllin, 40 ~./mL of streptomycin, and 10~
FCS. The cells axe a gift from Dr. Gazer, Texas A & M.
Endometxial tissues (approximately 600 mg wet
weight/culture dish) from Day 1S cyclic ewes were cultured
in 20 mh o.f DMEM supplemented with 40 units/mL of
penicillin and 40 ~ /mZ of stxeptomyc~,n, wh~.ch were treated
with 102 IU/mL of rhIFN-a , xhzFN~-y , ox rbzFN-~ . The doses
of those TfNs had been determined by the dose-response
experiments with monocytes. After 20 hours at 37°C in a 5$
Co2 atmosphere, culture medXa and endometxxal t~,ssues were
frozen separately and stored at ~70°C until subsequent
Western and Northern blot analyses.
[Clon~.ng of Ovine zP--1.0]
Complementary DNA library construction and
substxuct~.on experiments were completed using Day 17
pregnant and Day 15 cyclic endometrial RNAs according to
the method descxxbed xn ~.kopyants N.S. et al., pros. Natl.
Aced. Sci. U.S.A., 95: 13108-1311.3 (1998). A cDNA fragment
encoding Ovirie I1~-10 was ~.deritifxed from numerous cDNA
substruction studies. The full~length IE-10 was
subsequently obta~,ned us~.ng 5~ -RACE, 3~ -RACE, and full--
length polymerise chain reaction (PCR) methodologies
(Kuraishi T. et al., (2000) Biochem. J., 347: 579-583).
The PCR produots of full-length IP-10 cDNR were ~.igated to
CA 02497497 2005-03-02
_ 69 ,
a pGRIITM vector (Invitrogen) and then subjected to an
automated sequence analysis using a pcrkin-Elmer aequencer
(model ABI Prism 377 XL: Roche Molecular Systems,
Branchburg, N,~, u.s.A.). Nualeatide sequence of ovine IL~-
l0 cDNA were analyzed with Genetyx software pxogxam
(Softwaxe Development Co., Ltd. Tokyo, fapan).
[Preparation of probe and semiquantz.tative analysis by RT--
PCR]
To prepare cRNA probes specific fox ov~.ne xkN-'c ,
IP-10, and G3PDH, respective cDNAs were generated fxom
o~Vinc cndomctrial or conceptus RNA using RT-PCR. Total RNA
samples were reversely txanscxibed with SuperScriptIIz"'
(Invitrogen) and oligo-dT primex (20 ~ h xeaction volume),
and the resulting cDNAa wexe then subjected to fCR
amplification with primers shown in Tabl~ I.
Table z
Name Sequence Length(bp)
ozP-~.o Foxwaxd 5' -CACTCCTGhACTGTTCF1GGG-3' 262
Reverse 5'-CCATTCCTTTTCATTGTGGC-3'
oCXCR3 Forward 5',GCATCAGCTTCGATCGGTAC--3' 2B3
Reverse 5'-GATGCGGGCGTAGCAATAGG-3'
oI~-z Forward 5'-CATCTTCCCCATGGCGTTCG-3' 603
Reverse 5'-TCATCTCAAAGTGAGTTCAG-3'
oTFN-'yForward 5'-CGATGAAATACACAAGCTCC-3' 504
Reverse 5'--GATTACATTGATGCTCTCCG--3r
oG3PDH Forward 5'-ATGGGGAAGGTGAAGGTCGG-3' 901
Rcvessm 5'-ATGTGGGCCATGAGGTCCAC-3'
Forward 5'-ATGGGGAAGGTGAAGGTCGG-3' 149
Reverse 5'-ATGTGGGCCATGAGGTCCAC-3'
Table I shows primer sets used for PCR. When~in,
Name: taxget gene's name fox PCR amplification, sequence:
nucleotide sequence of primer, and Length: chain length of
the taxget gene_
CA 02497497 2005-03-02
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Eaah RT-PCR-derived fragment was subcloned ~.nto
the pCRIITM vector and then subjected to an automated
xequence analysis. Sequence compax.isons were performed
using the BLAST network program (National Center for
BiotechnoJ.ogy Information, NIH, Bethesda, MD, U.S.A.) and
confirmed correct ovine cDNAs. DIG-labeled cRNA probes for
Northern blot, analysis were generated from those cDNA
constructs using T7 RNA polymerase or SP6 RNA polyrnerase
(Kuraishi T. et al., (2000) Biochem. J., 3A7: 579-583).
Amounts of uterine IFN- y mRNA and CXCR3 mRNA were
determined from PCR amplification using oligonucleotide
primers (Table z). Each reaction consisting of primer
pE~irs for IfN- y /G3PDH or CXCR3/G3PDH was run with RT
template ( 1 a L ) and AmpX~.Taq Gold T" ( 1. 25 UJnit; Roche
Molecular Systems) in a final volume of 25 ~cz,,. Ration of
primer pairs that gave each PCR product within the linear
xarige haci been determined, i.e. 6:1 for IFN- y:G3FDH and 5:2
for CXCR3:G3$DH. pGR conditions were 95°C for 11 min, 40
cycles at 95°C for 1 min, 57°C for 1 m~.n, and~72°C for 1
min followed by a fxnaJ. extension at 72°C for 5 rnin. PCR
products were quantified using an image analysis system
(ToYoBo, Oaaka, Japan) equipped with Quantity one v3.0
s o f twa~re .
(Northern blot analysis]
Total. RNA was isolated from some endometrial and
conceptus tissues or cells using Isogen (Nippon Gene,
Tokyo). Poly(A)"RNA was abtairied from total RN1~ that had
been isolated from PBMCs (TaKaRa, Tokyo, Japan). These
RNAs were separated by electrophoresis on a 1_p$ agarose-
formaldehyde gel and transferred to a nylon membrane
(Bxodyne-8: Pall, East Hills, NY, V.S.A.). The nylon
membrane was prehybridized in the hybridization butfer
containing 5x SSC, 50~ foxmami.de, 50 mM PBS, 7~h SDS, 0.1'b N-
lauryl sarcosine, 50 ug/mL salmon sperm DNA (ssDNA), and 2~
blocking reagent (Roche Diagnostics, Mannheim, Germany) at
65°C for 1.5 hours, and then hybridized with cRNA probe in
CA 02497497 2005-03-02
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fresh hybxadization buffer at 63°C for 12 hours. AftQr
hybradazati4n, the nylon membrane was washed price with 2x
SSC and 0.1~ SDS at 65°C for 30 min, washed twice with 0.1x
SSC and 0.1~ SDS at 65°C fox 30 min, and then incubated
with RN~xse-A (20 ;cg/mL) at 37°C for 1 hour. The nylon
membxane was incubated in the blocking buffer (l~ blocking
reagent) at zoom temperature for 1 hour followed by the
addition of anti--DZG antibody (1:10,000 dilution: Roche
D~.agriastics, Mannheim, Germany). The pylori membranm was
treated three tames with the washing solution containing
100 mM malefic acid (pFi 7.5), 150 mM NaCl, and 0.3~ Tweeri 20
for 10 min each and finally rinsed in 200 mM Tri-HC1 (pH
9.5) E~rid 100 mM NaCl. The chemlumanescent reaction was
performed in 100 mM Txa-HCl (pH 9.5) and 100 mM NaCl
aoritaining CSPD reagent (1:200 dilution; Roche Diagnostics,
Mannheim, Germany), and the nylon membrane was exposed to
X--x~xy film.
[Iri situ hybridization]
In situ hybxadizatiori was performed according to
a known method (Kanai Y. et al., J Cell Siol., (1996) 133:
667--681) with minor modification. Frozen tissues were
sectioned (10 ~.m), mounted onto silan-coated slides, and
fixed in 4R~ paratormaldehyde in PBS. Sl3.de sections were
pretreated sequentially with 0.2 N HCl z~nd 20 ~cg/ml
proteinase K in Tris--HC1 (ph 7.6), Q~s paxafoxmaJ.dehyde, and
D.2~ gzyc~.ne. Slide sections were then prehybridized in
the solution Containing 50~ formamide, 5 x SSC, 1 x
Denhaxdt~s, 100 ~cg/m~ heparin, 10 mM dithiothreitol, 10'b
dextrin su~.fate, and 0.1 mg/mIr denatured tRNA and ssDNA,
followed by hybridization with DIG-labeled antisense or
sense cRNA pxobes at 45oc fox 18 hours. After
hybridization and washing once with ~! x SSC at 42°C for 20
min, slide sections were incubated with RNase-A (10 a g/mL)
at 37°G for 30 man and then wz~shed twice 2 x SSC apt 65°C fox
30 min and twice with 0.1 x SSC at 65~C fox 30 min. The
slides were blocked with a blocking reagent (Boehririger
CA 02497497 2005-03-02
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Mt~nnh2im) and incubated with an anti,DIG alkaline
phosphatase-conjugated antibody (Boehringer Mannheim). The
signals wexe detected with 5-bromo-4-chloro--3~-indolyl
phosphate and nitro blue tetaozlium (Promega, Mad~.son, Wx,
CT . 8 . fir, . ) .
[Expression and purification of recombinant caprine IP-10]
Thxough the use of RT-PGR, caprine IP--1.0 ( cIP-10 )
cDNA was amplified from caprine endometrial RNA by using
primer set as in ovine IP--10. The cDNA was cloned into a
pSTBlue (trade name; TaKaRa) plasmid. The nucleotide
sequence encoding the mature xegion of crP-10 was amplified
and c7.oned into an expression vector, pET-14b (trademark,
Novagen, Madsion, WI, U.S.A.), which consisted of a
hi.stidine tag at the N-terminal side of cIP-10. The
resulting expression vector was referred as pEx-7.4b-cZP-10.
The expression vector was used transform Escherichia coli
BL21-SI (Invitrogen) cells. The grown cells were harvested,
suspended ~.n 50 mM I~aF3zfOq, 500 mM NaCl, and 10 mM imidazole
(pIi 7.4), and disrupt~d on ice by sonication. After cell
debris and insoluble proteins were removed by
aontrifugation, recombinant czP-1.Q in the supernatant was
examined using SDS-PAGE. Recombinant cTP-10 was purified
us~,t~g a nickel-chelating column (Hi-trap Chelating HP,
Amersham Pharmacies 8iotach) on the chromatography system
(AFtTA, Amersha~m Pharmacies Biotech). The protein was eluted
from the column with a linear gradient of imidazole (20500
mM), which then dialyzed against P$s to remove imidazole.
[Western blot anaJ.ysis]
The culture media after in vitro culture of
endomotrial tissues and the dialyzed protein (recombinant
zP-~~.o) were analyzed far the pxesence a:~ xQ-10 by using
Western blot analysis. Culture medium (40,u ~.) or
recombinant IP-10 (50 ng/40 a L ox 200 ng/90 a L) was boiled
fox 5 m~.n in the SDS sample buffer, e~.ectxophoresed on 15R~
SDS-PACE gels undEr reducing conditions, and transferred
CA 02497497 2005-03-02
_'
onto nitxocellu7.ose membranes (Immunobilon: Millipore,
Bedford, MA, U.S.A.). The membranes were bJ.ocked with
Slack Ace (Dairiippori Pharmceutical, Osaka) at room
temperature for 1 hour and then incubated with a mouse
monoclonaX antibody to human TP-10 (Genzyma/Techne,
Minneapolis, MN, U.S.A.) or with a rabbit polycXonal
antibody (Sigma) to histidinw tag at 4°C for 12 hours.
After incubation, tho membranes were washed three times (10
min each) in a TBS-Tween 20 solution, and incubated with
donkey anti-mouse ZgG ox anti-rabbit IgG conjugated with
horseradish peroxidases at room temperature for 1 hour, and
washed three times (lg min each) in a TBS-Tween 20 solution.
The bands were detected with SuperS:~gnal Walt Femto Maximum
Sensitivity Substrate kit (Pierce, Pockford, IL, U.S.A.).
[Immunohistochemical analysis]
Whole ovine uteri containing bath the endametrial
and conceptus tissues were fixed in 4~ paraformaldmhyde,
embedded in paraffin, arid slide sectioned (6 a m). After
deparaffin, slide sections were immersed in 0.01 M sodium
citrate buffer solution (pH 6.0) and treated in autoclave
at 121°G for 15 min. ThE slide sections were incubated in
a methanol solution containing 4.5~ H202 at room
temperature for 1 hour, then incubated with Block Ace in a~
high humidity chamber for 1 hour. Then, the slide sections
were incubated w~.th a first antibody, i . a . axxt~.-human x~~-14
monoclonal mouse antibody (1 ,u g/ml: Genzyma/Techne) or
polyclonal anti-human IFN--y (500 ng/ml: Peprotech, London,
U.K.) at 4°C overnight. After the incubation, the slide
sections were treated with n secondary antibody (vectastain
RBC Kit: Vector Labs, Burlingame, CA, U.S.R.) for 1 hour,
and then incubated w~,th an avidin~biotin complex in a PBS
at loom temperature for 30 min. Antibodies bound to the
sZ7.de sectioYts were v~.s~uaZxzed with Metal. Enhanced DAB
Substrate kit (Pierce). For evaluating non-specific
binding (negative control), normal mouse or rabbit IgG
prepared to the same concentration ~.s used as semi-serial
CA 02497497 2005-03-02
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segments of tissues. The tissue segments were
counterstained with hematoxylin.
[~wc~'itro chemota~xis assa~y3
Migration of PBMCs was assessed in a 96-well
modified Boyden chamber (NeuxoProbe, Cabin ,john, MD,
U.S.A.) using polyvinylpyrrolidone--free polycarbonate
membrane (5 ~cm pore size; Neurohrobe), which had boars
coated with 20 ,ccg/ml bovine plasma fibronectin for 2 hours
before use. The assay was performed as described in
Gaspexini S. et al., (7.999) J. z~nlnuno~,., x.62: 4928-4937
with minor modifications. DMEM without FCS and DMEM
containing recombinant rP-10 (0.2 to 500 ng/mL) or the
supernatants from endometrial cultures treated with or not
treated with ZFN-T were added to the bottom we~.~.s of the
chemotaxis chamber, and PBMCs (5x106 cells/mL) in DMEM
without FCS were added to the top weJ.l.s of the chamber.
Rfter the chambers were incubated at 37°C in a 5~S
atmosphere for 2 hours, the membranes were removed, washed
with PBS, fixed, and stained with Did-Quick. The number of
ce~.ls that m~.gxated to the 7.owex layer was microscopically
counted in six randomly chosen high--power fields. Por the
blacking experiments, the supernatants from endometxium
stimulated with IfN-t or 5 ng/mL of recombinant ZP-10 were
preincubated at 37°C for 1 hour with 30 ~cg/mL of anti-=8-10
or control. mouse IgG before addition to tha top ck:ambex_
The assays were replicated in triplicate.
[statistical Rnalysix~
Each data point shows three samples that are
independent to each other and represented by means ~ SEM_
xhese data were ana~.yzed by one-way ANOVA, and then
Duncan's multiple range tests. Rddit~.onal,ly, Northern
blotting data in pregnant ovine were analyzed by regression
analysis using the ~.ea~ct-squares method. Differences with
a P values C 0.05 were considered statistically significant.
CA 02497497 2005-03-02
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Results
[Cloning of IP-10 cDNA]
Ovine IP-IO cDNA fragments were obtained from
endometrial tissues collected on Day 7.7 of pregnancy, and a
full-length ovine zP--7.0 CDNA was cloned using S'-RACE and
3'-RACE. The resulting ovine IP,10 cDNA encompasses 1172
base pairs {bp) with an open reading frame (ORF)
corresponding to x.02 amino acids. The nucleotide sequence
of the cloned ovine TP--10 and an amino acid sequence
deduced therefrom are shown in fig. 1. In 1172 by cDNR
sequence (GenBankz'"i Accession No. AB070717), shown in Fig. l,
containi.rig the ORF corresponding to 102 amino acid residuary
the first 9 amino acids segment of the coded amino acid
sequence {ovine rP-10: GenBankTM Accession No. BAB63958)
was presumed as a signal peptide.
Capxine xP-10 cDNA was cloned from caprine
mndom~trial RNA by RT-PCR using a pxx.mer set designed from
ovine IP-10 cDNA. The cIP-10 cDNA sequence was the same as
that of caprine other than that the coded 99th amino acid
residue was axginine in cIP-10 instead of glutamine in
ovine IP-10.
Comparat~.ve ana7.y$is of the amino acid sequences
among various species is shown ~.n Fi.g. 2. Four cysteine
r~sidues are conserved within chemokine family, and the
first two cysteines are separated by a single amino acid to
make C-X-C. This cysteine motif wasp found xn amino acid
residues deduce from ovine IP,10 cDNA and caprine IP-10
cDNA. The C-X-C chemokines axe subdi~crided into two classes
depending on thQ presence of the glutamin~-leusine-arginine
(ELR) motif preceding the first two cysteines (Rollins H.J.,
(1997) Chemok~.nes, 90: 909-928). Similar to IP-lOs of
other species, the ovine IP-10 lacked the ELR motif and
showed a high degree of similarity '~o human ZP-10, i.e.
92.75 and 75.5R~ at the nuc~.eotide and amino said levels,
respectively (Table II).
CA 02497497 2005-03-02
Table II
Nucleotide Sequence (~)
Ov~.ne Caprine Human Mouse
Ovine - 98.7 82.7 75.2
Caprine 98.0 - 82.3 '75.2
I~uman 75.5 75.5 - 74.4
Mouse 67.3 67.3 67.3 -
Table Z1 shows the homology (identity) among
ovine, caprine, human, and mouse in amino said and
nucleotide sequences.
[Changes of ZP-10, CXCR3, IFN-z, and IFN-y mRNA expression
during early pregnancy]
Uterine IP-10 mRNA levels on Days 14, ~,7, 20, 25,
and 30 of pregnancy were examined by Northern blot analysis
(Fig. 3). Fig. 3 shows levels of zP-3.0 mRNA expression in
the ovine uterus during early pregnancy. xn left of Fig. 3,
results in IP-10 m~iNA in the uterus of pregnant ewes (n = 3
for each of Days 14, 17, 20, 25, and 30), cyclic ewes (Day
15, n = 3), and in the Day 17 conceptus (Con) are shown,
which are each one result from three independent
exper~.ments. xn right of Fig. 3, signal tatios~ =P-10/G3PDH
derived from all the results of Northern blot analysis of
Ip--7.0 axis rxhown, in which mean ~ SEM are represented by
bars. An asterisk in right of Fig. 3 indicates a
statxatiCal difference (p < 0.05) when compared with the
value from Day 15 cyclic uteri.. Result of xcgxeas~.on
analysis on IP-10 mRNA and day of pregnancy was y = -51.5x2
+ 288.2x ~ 131.2 (R2 - 0.823, p < 0.01).
Consistent with the result Pram ovine rp-10 cDNA
cloning experiments, a single transcript (approximately 7..1
kb) for IP-~10 mRNA was detected in uterine endometrium of
progttant awes. The expression of endometrial IP-10 mRNA
was much hi.ghex on Days 7.4, 17, 20, and 25 of pregnancy
CA 02497497 2005-03-02
than that in cyc~.xc ewes. Expression of endometrial cXCR3
mRNA, a recEptor for IP-10, was higher on Days 17 and 20 in
pregnant ewes (Fig. 9). fig. 4 shows lev~1s of CXCR3 mRNA
expression. In left of Fig. 4, resuXts of semiquarititative
PCR of CXCR3 mRNA and G3PDH mRNA in the uterus of pregnant
(n ~ 3 for each of Days 7.4, 17, 20, 25, and 30) and cyclic
ewes (Day 15, n a 3) are shown, which are each one result
from three independent experiments. In xi.ght of Fig. 9,
results of densitametric analysis of semiquantitative PGR
products for CXCR3 mRNA and G3pDH znRN,F. are shown as ratios
of CXCR3 mRNA to G3PDH mRNA (CXCR3 mRNA/G3PDH mRNA). Bars
represent mean t SFM. An asterisk in right of Fig. 4
~.ndi.catos a statistical difference (p < 0.05) when compared
with the value from Day 15 cyclic uteri.. ResuJ.t of
regression analysis on CXCR3 mRNA and day of pregnancy waa
y = -8 . 9x2 + 45 . 8x ~- 61. . 4 ( R2 = 0 . 931, p < 0 . 01 ) .
Fig. 5 shows l.evel,s of ZFN- z and IFN- y mRNA in
the ovine conceptus and uterus during early pregnancy. zn
left (A) of Fig. 5, results of Northern blot analysis of
IFN-z mRNA in the Gonceptuses of pregnant ewes (n = 3 for
each of Days 14, 17, and 20) axe shown, a;nd in right (S),
results of s$miquantitativ'e PCR of IFN- y mRNA and G3PDH
mRNA in the utexws of pregnant ewes (n = 3 for each of Days
7.4, 17, 20, 25, atnd 30), cyclic ewes (Day 15, n = 3), and
in the Day 17 conceptus (Con) axe shown, which axe each one
result from three 5.ndependent experiments. In the bottom
of right in Fig. S, results o~ densitometric analysis of
semiquantitative PCR products of IFN- y mRNA and G3PDH mRNA
are shown as ratios of IFN- y mRNA to G3PDH mRNA (IFN- y
mRNA/G3PDH mRNA). Bars rmprmsent mean t SEM. An astmrisk
indicates a stat~.st~.cal difference (p < 0.05) when compared
with the value from Day 15 cyclic uteri. Result of
regression analysis on IFN- y mRNA and day of pregnancy was
y = -10.6x2 ~ 56.6x -~ 81.3 (R2 = 0.762, p < 0.01).
Expression of IFN--r mRNA was detected in the
conceptus, and the level of this expression was higher in
the Day ~.4 ooriceptus than others (Fig. 51~). Expression o~
CA 02497497 2005-03-02
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IFN- v mRNA was detected in the uterine endometrium of bath
pregnant and cyclic ewes and was higher on Days 1,4, 17, 20,
arid 25 in pregnant ewes that ethers (Fib. 5H).
[Localization of IP,10 and IfN- y proteins]
fo determine localization of IP,10 and zFl~~- y ,
immunohistochemicaX analysis was performed on sections of
uterine and conceptus tissues prepared from Day 13 cyclic
and Day 1~ pregnant ewes (Fig. 6). Tn Fzg. 6, the Day 15
cyclic ewes (Left; A, B, and C) and Day 17 pregnant ewes
(right: D, E, and F) are practically shown as a series of
sectiaris. Photomicrographs 1~1 and D Shaw xP-10, B and E
show IFN- y, and C and F show control.
Both IP,10 and IfN- v proteins localized in
luminal and glandu7,ax epithelium and subepithelial stroma
of the cyclic and pregnant uteri. Amount of IFN- v protein
detected in endometxium of GyaliC ewes was the same as that
of pregnant ewes (Fig. 6). IP-10 protein was detected in
endometrium of cyc7.~,c ewes, but the degree of the staining
was the mi,ni.murn.
[Localizatian of TE-10 mRNA]
To determine a cellular source of zp~-1,0 j.ri the
sheep endometr~.um, xn situ hybridization was performed on
sections of uter~.ne tissues prepared from l7ay 15 cyclic and
Dsy 17 pregnant ewes (Fig. 7A). In Fig. 7,
photomicrographs a and b are xesul.ts of analysis of IP-10
mRNA in the uterus xn pay 17 pregnant ewes by DIG-labeled
antisense IP--10 cRNA, photomicrograph c is resu~,ts c~f
analysis of 2P-10 mRNA in the uterus in l7ay ~.7 pregnant
ewes by DzG-labe~.ed sense IP-10 aRNA, and photomicrograph d
is results of analysis of I8-10 mRNx1 ih the uterus in Day
15 cyclic ewes by DIG-labeled antisense IP-1.0 cRN~I.
IP-10 mRNA was detected in the subepithelial
stroma but not a.n the luminal and glandular epithelium of
the pregnant uterus. At higher magnification, the signal
appeared to be present in immune cells. Northern blot
CA 02497497 2005-03-02
analysis was performed to identify tissue and/or ce~~ types
expressing IP-10. TP-10 mRNA was found in RNA extracted
from monocytes but not in RNA from lymphocytes, epith~lial
Dells, or stroma cells (Fig. 7B).
[Effect of IFN-z on IP-10 expression]
Various IFNs weze investigated for their dose
responses and their ability to stimulate IP-10 mRNA
expression in monocytes. Monocytes isolated from ewes were
cultured in vitro for 24 hours in the presence ox e~bsence
of ~.Oz to 10° IU/m~ of IFN- a , IFN- y , or IFN- ~c . Effects of
several, xFNs on IP-10 mRNA levels are ahowri in Fig. 8.
Results of the dose-response experiments for the expression
of IP-10 mRNA in IFNs-stimulated monocytes axe shown in Fig.
8A, which are each one result from three independent
experiments.
All IFNs stimulated the expression of IP-10 mRNA
in ovxt~e monocytes, however, the effective doses Were
different fox each IFN (Fig. 8A). IFN-r at a dose of 1,02
IU/rnL stimulated the expression of IP-10 mRNA mere
e~~ectively than the other IFNs examined.
In cyclic ewes, it was investigated whether IFNs
could effect endometrial =P-10 mRNA production.
Endometrial exp7.ants from Dmy 15 cyclic ewes were cultured
in vitro far 20 hours in the presence ox absence o~ lOZ to
x.04 IU/mL of 2FN- rx , IFN- y , or xfl~- z . Total RNA was
extracted from IFN-stimulated endometxisJ. tissues and
examined fox x~e-is mRNA by Northern blot analysis. Minute
levels of IP-10 mRNA were detected in controls and in IFN-
a -stirnu~.ated ox rFN- y -stimulated saznple$, but high levels
of xP-10 mRNA ware found only after stimulation with zFN-s
(Fig. 88). Western blot analysis confirmed that
endometrial IP-10 production was stimulated by IFN- ~ (Fig.
8G).
Fig. 8B shows effaat of several IFi~s an rP-10
mRNA levels in the endometxia~. eXp7~ants from cyclic ewes.
In left of Fig. 8B, results of Northern blot analysis of
CA 02497497 2005-03-02
-$(f_
IP,1Q mRNA in the endometrial expJ.ants stimulated by 10z
Zt~/mL of IFN-a , TFN-'y , or IFN- z are shown, which axe e2~ch
one result from three independent experiments. In right of
Fig. 8B, xesults of densitometrie analysis of Northern
blots of IP-10 rnRNA and G3PDx mRNA are shown. Signal,
ratios xk~-14/G3PDH were derived from a11, the results of
Northern blot analysis. Bars represent mean t SEM. An
asterisk indicates a statistical difference (p < 0.05) when
compaxed faith the value from endometrial, e~tplant.s cultured
without IFN. In Fig. 8G, results of Western blot ana7.ysxs
of pxoteins in the culture medium fxom ez~dometrial explants
stimulated by IFNs are shown.
[Effect o~ x~e-is on PBMC migration]
To ascextaj.n whether recombinant caprine TP,10
(xczP-10) and endometrium-derived ZP-10 were biologically
active, effect of rcTP-~.0- or rFN-t-stimulated endometrial
culture media on migratory activities og PBMCs were
investigated by in vitro chernotaxis assays. Recombinant
cIP~IO was puxified using a nickel chelating column, and
detected by Western blot using antibody against IQ-~.0 and
hist~.d7.ne-tag (Fig. 9A). Receptor for IP-10, CXCR3, mRNBi
exists in PBMC was confxxmed (fig. 9$, lane T: total RNA,
~c g, lane M: poly (ia) + RN.&, 2 a g ) . Chemotaxis assay
confirmed that f8NlC responded to 0.2 to 5 ng/mh of rcIP-20
but the reaction decreased in higher levels of rcTP-10. As
a result, the dose-response showed a characteristic belJ.-
shape curve (Fig. 9C). Medium fxom e~n.dometrial tissue
cu7.tuxe that had been stimulated by ZFN-r exerted
s~.gn~.ficant chemotz~xis effect on P$MCs, whereas the medium
abtained from the endometrium cultured without IFN-z was
ineffective (Fig. 9C). Bars represent mean t SEM. An
asterisk indicates a value (p < 0.05) when compared with
the migration of PBMCs with IFN-r-untreated endometrial
cuxtuxe medium. trouble asterisks indicate a ~sralue (p c
0.05) when compared with the migration of PBMCs in the
absence of rclP-10. Immunoneutralization experiments
CA 02497497 2005-03-02
- ~1 -
revealed that an anti-IP-~.0 ant~.body reduced the
chemotactic activities of culture medium from TP-10-- or
IFN-i-stimulated endometrium by 60 to 70~, xndiaating that
the chemotactic activity exhibited by the culture medium
from IFN~'c-stimulated endometsium was due mostly to TP~10
(Fig. 9D). Bars represent mean ~ SEM. An asterisk
~.~tldiCates a migration value of PBMCs in TFN- s ~~txeated
endometrial culture medium in the absence o~ an anti-IP-10
antibody at p < O.OS. Double asterisks indicate a
m3.gration value of PBMGs by IP-10 in the absenca of anti-
TFN-r antibody at p < 0.05.
~Disaussionl
The present invention disclosed that ovine IP-1.0
Was present in the pregnant uterus and to a much lesser
degree in the cyclic endomctrium. ZP-10 is a member of th~
C-X-C chemokine family, which regulates multiple aspects of
inflammatory and immune responses through the recept,ox for
IP-10, CXCR3, and/or chemotaGtiC activity of subsets of
leukocytes (Earber J.M. et al., (1997) J Leukoc Biol-, 61:
246-257).
The present invention successfully disclosed by
the dose-response experiment us~,ng monoaytes that IFN- a,
xFN- y, and IFN-r stimulated IP-10 mRNA express~.on, but the
effective doses differed among zFNs. IfN-s effectively
stimulated IP-10 expression in monocytes at a dose of 10Z
IU/mL, whereas xhzk~l- y at the same dose was less effective.
It is unclear whether this low level of stimulation of IP-
mRNA by zFN- y reflects the fact that IFN- y is highly
species specific cornparad other IFNs (pes~tka S. et al.,
(1987) Ann. Rev. Biochem., 56: 727-777). Tt should be paid
attention that a high dose of rhIFN- y (10° Tc7/mL) stimulated
the expression of TE-~.0 mRNA in monocytes. Thus, the low
stimulatory effect of IFN- y on TP-10 production was nOt
solely due to a Ross of activity. The pattern of
endometrial IP--10 mRNA expression appeared to have fol~.owed
CA 02497497 2005-03-02
- 82 -
that of conceptus IFN-t mRNA during early pregnancy, and
Changes in IP-10 exgression were similar to changes in
other endametrial chemakines sucri as monacyte
chemoattractant protein (MGP)-1 and MCP--2.
IP-~,D mRNA was detected by ~.n s~.tu hybridization
analysis in the subepithelial stsoma, and the signal
appeared to be localized in immune cells. zn studies about
human and mouse hitherto known, TP~lo is secreted by
monocytes (Luster A. et 'al., (1985) Nature, 315: 6'72-676
ohmori X. et al., (1990) 8iaahem. Siophys. Res. Cammun.,
168: 1267.-12&7 ) , and xn the present ~.x~,vez~tiari, rP-10 was
likewise expressed in ov~.ne monocytes but not lymphocytes,
epithelial cells, or stroma cells. MCP mRNAs are also
localized in the subcpithelial stroma, but MCP-positive
cells are eosinophils (Asselin E. et al., (2001) Biol.
k~eprod., 69: 992-1400). These observations suggest that
zP-10 in the subepithelial stroma is produced by resident
macrophages and/or m~scxophs,ges that have been recruited to
that region. IP-10 protein was localized in luminal and
glanduJ.ax epi.the~.xum of the uteri as well as l.ocal~.zed in
subepithelial stroma region. In the conceptus, very small
amount of zp-10 protein was detected, but zk~-7.0 mRNA was
not detected. It was suggested that IP-10 produced by
macrophages in the subepithelial region mxgxate into
epithelium, which stimulates migration of CXCR3-expressing
immune cells toward or near the site of conceptus
implantation to the maternal endometrium.
As the conclusion, the present invent~.on revealed
that IP-10 and IfN- y mRNA expressions time-~craryingly and
cell-specifically occur during early pregnancy in the ovine
uterus and that concep~,us TFN-s has an ability to regulate
endometrial TP-10 expression. These fa.ndings suggest that
IP-10 regulated by IFN-s plays an impastant role in the
recruitment and/or redistribution of immune cells during
early pregnancy in ruminants.
CA 02497497 2005-03-02
'Ihe present invention pxovides a way for
investigating relationship between ZP-10 and IFN-r and Ip--
functions ~.n uterus.
Example 2
fQxeparation of animal tissues]
Uteri fxom cyclic goats on Days 14 and 17 and
pxegnant goats on Days 14, 17, and 2Q wexe xemaved by
hysterectomy undex ~.soglurane anerthesis. Whole uteri from
Day 14 cyclic and Days 14 and 17 pregnant goats were frozen
fvr in situ hybridization analysis. UterJ, fxom Days 14 and
17 pregnant and Daya 14 arid 17 cyclic goats were subjected
to uterine flushing procedure for the iso~.ation of
conceptuses and/ox aalleation of flushed media. PBS (15
was gently flushed into the uterus, and the utexine
flushing media collected wexe concentxrsted (Macrosmp, Pall
Gorposation, East Hills, NY) and subjected to the Westexn
blot analys~.s fox the detention of Zf-10. Gonceptus
tissues collected from Days 14 (whole conceptus), 17
(approximately one half of conceptus), arid 20 (trophoblast
rec~a.or~ only) of pregnant goats were subjected to in vitro
culture. The remaining Days 17 and 20 conceptus tissues
wore frozen for subsequent RNA extraction and
immunostain~.ng . The ~.n v~.txo cu~.ture media were sub j acted
to Western blot analysis for the detection of ZFN- z. Uteri
fxom Day 14 cyc~,zc goats wexe used for ep.~thez~.al cell
isolation. Endometrial explants from Day 1A cyclic goats
were cultured to examine the stimulatory effect of IFN-s on
xP-10 production. Eridometrial t~.ssues of goats from which
conceptuses had been xemoved were frozen and subjected to
RNA extraction.
(In vitro culture]
Whole conceptuses (Day 14, n s 3) and minced
conceptuses (Days 17 and 20, about 200 mg/cultuxe dish, n =
CA 02497497 2005-03-02
-$4-
3 each) were cultured under conditions according to a
method of Imakawa, et al.'(Endocrine (1995), 3, 5J.1-517).
The procedures for endometxi~xl epithelial cell
dissociation and subsequent characterization wexe performed
according to a method of Takahashi, et al. (J. Reprod. Dev.
(2001) 47, ~,8z-187). Uterine horns from Day 1.4 cyclic
goats were filled with o.'76R~ ETNA-PBS and preincubated at
37°G for 1 hour. The endometr7.~x1 epithelium was scraped
off by a surgical blade and then incubated at 37°G for B
min in a 0.1~ colXagenase (Sigma, St. Louis, MO). The cell
cusp~nsion was passed through a nylon mesh (70 ~tm) and
subjected to density gradient centrifugation. Co7.J.ected
epithelial cells were resuspended in DulbeCCO's modified
Eagle's medi.um/Ham's F-12 (DMEM/Ham's F12, Sigma)
supplemented with 40 units/mL of penici~.l~.n, 40 ~cg/mL of
streptomycin, and 10~ FCS. The cells were plated in 24-
well dishes coated with sw~.ne-skin type x collagen (Nitta
gelatin, Osaka, Japan) and subjected to the adhesion assay
whon the cells had grown to confluent.
Cells were prepared from Day 17 goat conceptus.
These conceptuses were dissected into small pieces and
incubated in 0.2~ collagenasE (Sigma) at 37°C for 30 min.
The cells were passed through a nylon mesh (70 ~cm) and
resuspended in DMEM with antibiotics, which were
immediately used for chemotaxis ox adhesxori assays. In
addition, bovine B cells (ICU-l, 33) and caprine trophoblast
clone cells (HTS-1, 33) were cultured in DMEM supp~.emented
with 10% to 20~ FCS and ant~.bodi,es.
Endometrial tissues (about 600 mg wet
we~.ght/culture dish} from Day 14 cyclic goats were cultured
in 20 mL of DMEM supplemented with antibiot~.cs, aced were
treated with 5 nM recombinant bovine zEN- r (rbIFN-s,
Katakuxa industries Co., xric., Tokyo, Japan). After 24
hours at 37°C and 3~ COZ atmosphere, endometrial tissues
were frozen and stoxed at -70°C for subsequent RNA
extraction and Northern blot analyses.
CA 02497497 2005-03-02
-$S-
[Cloning of capxiria IP-10 and CXCR3 cDNA]
Through the use of RT-PCR, ss shown Exarmple 1,
caprine xP-10 and CXCR3 cDNAs were amplified from capx3.ne
~ndometrial RNA and subc~.oned into a pSTBlue vector (Takara,
Tokyo, Japan), which was then subjected to an automated
sequence analysis using a PerkinElmer sgquencer (model ABI
Erism 3~7 XL, Roche Molecular System, Branchbuxg, NJ).
Nucleotide sequence comparisons of caprine TP,~.O and CXCR3
wexe performed using the BAST network program (National
Center for BiotechnoXogy xnfoxrnatipn, NIH, Bethes~da, MD)
and GenBank accession numbers were obtained (AB099892 and
AB099B93, xespactivaly).
(Transfection of CXCR3 expxess.ion p~,asm7.d]
The nucleotide sequence encoding the open reading
frame of caprine CXCR3 was PCR-amplified tend alaned into a
mx~mmalian expression vector pcDNA3.1 (Invitrogen Corp.,
Carlsbad, CA). This plasmid was transfected into KU-1 and
HTS~-1 cells by uacing Transfr~stTM (Pramaga, Madison, Wz)
aacordint to the manufacturer's protocol. Two days after
the transfectzon, ce~.~.s wexe used for chemotaxxs ox
adhesion assay.
[Production of recombinant caprine IP-10 and the antibody]
The nucleotide sequence encoding the mature
region of caprine IP-10 (cTP-10) aDNA was ECR-amplified and
c~,oned into ari expxesss~,on pl,asmid, pET-14b (Novagen,
Madison, WI), which consisted of a histidine tag at the N-
terminal side of cIP-10. This expxdss~.on vector, pET--14b,
cIP-10, was used to transform Escharichia coli BL21-SI
(Invitrogen) cells, which were cuXtuxed overnight. These
cells wcxe harvested, resuspended in 50 mM NaHZPOg, 500mM
NaCl, and 10 mM imidazole (pH 7.4), and disrupted on ice by
sonication. Rfter cell debxi.s and insoluble proteins were
removed by centrifugation, recombinant crP-10 (rcx8-10) in
the supernatant was examined us~.ng SDS--PAGE. Recombinant
cIP-10 was purified using a nickel-chelating column (Hi-
CA 02497497 2005-03-02
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trap Chelating HP, Amersham Pharmacies Biotech Znc.,
Buckinghamshire, UK) on the chromatography system (AKTA,
Amersham Pharmacis Biotech Inc.). xhe protein was eluted
fxom the column with a linear gradient of imidazole (20 --
500 mM), which was then dialyzed against PBS to xemove
imidazole.
Racombil'~al7t cxP-10 (200 ,~.c g) proteins in PBS were
mixed with Freund's complete adjuvant (Sigma) and anjected
subcutaneously into rabbits (n ~ 2). Recombinant cIP-10
proteins were injected two more times biweekly. A tatex
was ch~cked 1 week after the last ~.t~je~ction, and blood was
collected on the following day. Anti-capxine ZQ-x0
antibody was purified from antiserum using Hitrap Protein G
column (Amersham Pharmaci.a Biotech Inc.).
(SDS-PAGE and Waster~'1 blot analysis]
After determining protein concentration us~.ng
Protein assay kit II (Bio-Rad, Hercules, CA), concentrated
uterine flushing media (3 to B ~ L, ~.0 ~ g of protein/lane,
n = 3 each), or recombinant IP--y0 (50 or 200 ng/lane) were
subjected to Western blot analysis according to the method
of Nagaoka, et al., (Biol. Reprod. (2003y 68t 1413-1421).
Protein samp~.es were boiled for 5 min an the SDS sample
buffer, electrophoresed on 10~ or 15g SDS-PRGE gols undor
reducing conditions. The gel was stained with Coomassae
B~.u~s. Proteins in the gel were transferxgd unto
nitrocellu3.ose memb7rarses ( rmmobilon, Millipore, Bedford,
MA ) fur Western blot analysis. The membranes ware blocked
with B~,pck AGe (Dainippon Pharmceutical, Osaka) at room
temperature for 1 hour, and then incubated with a rabbit
polyclonal antibody to ovine ~FN-s 0,:5000, a gift of Dr.
Bazer), histidine tag (Sigma), ox aaprine IP-10 (1:1000) at
4°C for 12 hours. After incubation, membranes were washed
three times ,an xBS-Tween E0, incubated with donkey anti-
rabb~.t IgG-conjugated with horseradish peroxidases
(Amex~sham Pharmacies Biotech Inc.) at room temperature for Z
hour, and washed three tames ~.r~ xBS-Tween 20. The bands
CA 02497497 2005-03-02
_ $'~ _
were detected using ECL Western blot detection reagents
(Amersham Pharmacia Biotech Inc.).
[RT~~CR analysis]
Total RNAs were extracted from endometrial and
conceptus tissues and cultured cells (n = 3 each) using
Isogen (Nippon Gene, Tokyo, Japan). Total RNA samples were
reverse,txanacribed with SuperScriptZZ (Invitrogen) and
o~.igo-dT primers (20 ~.L of reaction volume). Amounts of
CXCR3, XCR1, and integrin receptor mRNAs were determined
from PCR amplif~.cation using oligonucleotide primers (Table
III) .
TabJ.a III
Name Sequence o~ forward and reverse Length(bp)
prime
CXCR3 5'-GCATCAGCfTCGATCGGTAC-3' 283
5'-GATGCGGGCGTAGCAATAGG-3'
XCR1 5'-ATGGAGCCCTCAGACATCCC-3' 627
5'-GAGGATCTCCACGTAGGAGA-3'
Integrin a5 5'-TGCTGTG.'AP.CCAGAGxCGTC-3' 80g
5'-ATCCACfGCACAGCTGTGGC-3'
Iritegrin 5'-GAAGCAGGAAAGAGAGCC'x'G-3' 890
aV
5'-CTATATCCGTGGCTCCTTTC-3'
Integrin X31 5'-CTCAAATCCAGCCACAGCAG-3' 523
5'-CCAGCGAAGTGAARCACAGC-3'
Integrin f33 5'-AGATTGGAGACACGGTGAGC~3' 392
5' -GTACTTGAAAGTGA'J.'CTTGC-3'
Integrin ji5 5'-GTCTGAAGATfGGGGACAGC--3' 2BS
5'-GGTACACGCTCTGGTTCTCG-3'
G3PDFI 5'-ATGGGGAAGGTGAAGGTCG6-3' 150
5',ATCATATTGGAACATGTAAA-3'
Each reaction consisting of primer pa~,rs was run
with RT tomplate (1 ~ L) and AmpliTaq Gold (O.GaS U: Raahe
Molecular System) in a final volume of 25 ,u L. All PCR
reactions consisted of 40 cycles at 95°C for 1 min, 60°C
Eos I min, and 72°C for 1 min, followed by the final
extension at 72°C for 12 min. After agarose gel
electrophoresis and visualization with ethzda.um bromide,
PCR products were qualitatively detected by using an image
analysa.s system (A'~TO Corp., Tokyo, Japan) equipped with
CA 02497497 2005-03-02
the Quantity One (v3.0 softwt~rt; PDI, Inc., Huntington
Station, NY).
[Northern blot analysis]
Northern blot analysis was performed as a method
of Nagaoka, et.al., (Biol. Reprod. (2003) 68: 1413-1421).
DxG-labeled cRNA probes were generated from xQ-10 and CXGR3
cDNA pJ.asmids by using T7 or sp6 RNR polymerase (Pxomega).
Total RNAs were used for the determination of IP-~.0 mRNA
whereas poly(A)+ RNA was used to examine CXCR3 mRNA.
Poly(A)~' RNA was obtained from total RNA that ha,d been
isolated from PBMCs using oligotex-dT30 (Takara). Total
RNAs (20 lcg/lane, n = 3 each) ox (A)+ RNA (2 a g/lane, n ~ 3
each) were separated by electrophoresis and transferred to
a nylon m~mbrane (Biodyne-B; Pall Co.rporation)_ fhe
membrane was hybridized with cRNA probe at 83°C for 12
hours, and then signals were detected using DIG detection
system.
(Tmmunof~.uorescence analysis]
Frozen conceptus tissues sect~.oned (10 um, n ~ 3
for each day examined) w~re mounted onto ailan-coated slide
and fixed in $cetorlo. Nonspecific binding was b~.ocked by
the treatment with Block Ace at zoom temperature for 1 hour,
and the slides were then incubated with a mouse monoclonal
antibody to human CXCR3 (10 ug/ml.R~D Systems Inc.,
Minneapolis, MN) of normal mouse zgG (Sigma) at 4 °C fax I2
hours. 3~fter the primary antibody incubat~.on, slides were
treated with fluoresceine isothioeyanate-conjugated goat
anti-mouse zgG (15 ~Zg/ml,Jackson ImmunoResearch
Laboratories Iria., West Grove, PA) at room temperature for
~, hour. Nuclei were stained with propidium iodide (2 l.~g/ml,
Sigma). The slidms were scanning examined and digital
images were captured on a confocal laser scanning
micxosCape (FV300, OLYMPUS, Tokyo, Japan)
CA 02497497 2005-03-02
.. gg _
[Protein biotinylation and detection]
Recombinant cIP-10 protein was biotinyXated by
BZ-link biotinylation reagent (Pierce, Rc~ckford, IL). As n
control, recombinant GST and xecembinant caprine
lymphotact~,n-C, ST protein were also labeled with biotin.
The biotinylation was performed by incubating these
protea.ns with 0.3 mg/mh EZ~l~.~nk biotinylation reagent (1
mL) in pSS ore ice for 2 hours. R,fter the incubation, the
resulting so~.utipn~ were dialyzed to remo~cte the free biotin
and measured their protein concentrations. The
biotinylated proteins (2 ~cg/l.ane) were appliod on 15~ SDS-
PAGE gels under reducing condition and ~cri.sualized by
strepav~.din-horseradish peroxidase (Amercham Pharmacia
Biotech Inc.) in order to confirm the labeling efficiency.
Frozen conceptus tissue sections (7.0 ~ g, n = 3
each) were mounted onto silart-coated slides and fixed in
acetone. The slides were blocked with Block Ace at room
temperature for ~, hour arid then incubated with biotinylated
protein (7.00 j,~g/mh) at room temperature xor 1 hour. After
the incubat~,on, the slides were incubated with
streptavidin-horseradish peroxidase (1:5000, Amersham
Pharmacia Biotech zz~c.) at room temperature for 1 hoax.
The biot~.ny7.ated protein bound to the tissues was
visualized with a DAB solution ($igma). Tissue sections
were then counterstained with hematoxylin.
[Chernotaxis assay]
Migration of KU-1, HTS-1, or primary trophoblast
cel.J.s were assessed in a 96-well modified Boydelt chamber
(NeuroProbe, Cabin Rohm, MD) using polyvinylpyrrolidone-
fre~ polycarbonate membrane (5 ~.cm pore size, NeuroProbe).
The polycarbonate membrane was coated with 10 ~cg/mL bovine
plaxrna fibronectin (wako ,Tunyaku, Osaka, sapan) in advance.
The assay was performed according to a method of Nagaoka,
et al. (Biol. Reprod. (2003) 6B: 1413-1427.). Nariteiy, DMEM-
0-Z~s SSA (without phenol red or FCS) supplemented w:Eth
indicated concentration of rclP-10 was added to the bottom
CA 02497497 2005-03-02
wells of the ahemotaxis chamber, whereas cells (5%106
cells/mL) ~.n DMEM-0.1.$ BSA (without phenol red or FCS) were
added to the top wells of the chamber. The chambers were
incubated at 37°C and 5$ C02 atmosphere for 2 hours, and
then the membranes were removed, washed with PBS, fixed,
and stained with pi~-Quick (Kokusai Shiyaku, Kobe, Japan).
The number of cells that migrated to the lower surface was
microscopica3.ly counted at six randomly Chosen fields. For
the blocking experiments, before addition to the bottom o~
the chamber, rclP-10 was preincubated with anti-IP-~.0
antibody or control rabbit IgG (Sigma) at 37°C for 1 hour.
Three independent experxme~lts were done for each treatment.
[Adhesion assay]
Twenty four-well plates were coated with type T
collagen (Nitta geJ.atin) or fibronectin at a concentration
of 10 ~cg/mh at room temperature for 2 hours, or plated with
caprine epithelial cells. Axter washing with PBS three
times, the plates were blocked with 1Rs BSl~ at room
temperature for 30 min. HTS-7. ox primary trophoblast cells
were labeled with the intracellular ~luoxesaent dye, 4 ,u M
calsein-AM (Molecular Pxobea Inc., Eugene, OR) at 37°C for
30 min. After washing With PBS three times, the cells were
incubated with the indicated rclP-Z0 at 37°C for 1 hour,
and were then added to each well. The plates werm
incubated at 37°C fox 7. hour, and then washed with PBS
three times to rmmove unbound cells. The remaining ceJ.7.a
were treated with PBS containing 1Rs Triton X-100 and 10~
ethanol. Fluorescence of cells was measured us~.ng
fluorescence reader (excitation filter 485 nm and emission
filter 535 nri1) {ARVO~M S?C 1420 Multilabel Counter,
Perl.inElmer Life Sciences Inc., Boston, MA). For the
blocking experiments, rcIP--~.0 protein was preiridubated with
30 a g/mL of anti--IP--10 antibody or control rabbit TgG
(Sigma) at 37°C for 2 hour. To investigate the inw'olvement
of IP-10 on cell adhesion to Fibronectin, the Gly-.fig-Gly-
Asp-Ser-pro--hys (GRGDSL°K, Sigma)synthetic peptide at a .
CA 02497497 2005-03-02
-41-
concentxation of 50 mM, its control, Axg-Gly-Glu-Ser (RGES,
Sigma), or 5 mM EDTA was preincubated with cells and rcxP-
7.0 protein. Three independent experiment were performed
for each substrate and treatment.
[Statistical analysis]
Measurement of optical density (Western blot) and
light intensity (RT-PCR) were subjected to least squares
(LS) ANC'vA, which employed the general linear models
procedure of the statistical analysis system (version 6.0:
SAS institute, Cary, NY). The light intensity from G3PDH
PCR products was used as covariates for RT-PCR analyses.
In chemotaxis assays, the number of cells migrated or
attached with a treatment was calculated as the number
xela~.ive to the cells without any treatment, which was then
analyzed stati.stxcally as aforementioned. The mode. ~xsed
in the Ls--ANOVA included treatment and replicate as sources
of variation. The least square means (LSM) and standard
error (S. E.) illustrated xn figures were derived from this
analysis.
[Preparation of rcl8-10 protein and the antibody]
Recombinant caprine TP-10 (rc=F~7.0) was expressed
in Escherichia eoli BL21-SI, which is transformed E. eoli
w~.th pET-14b-cIP-10 plasmid. The xcxP-10 was purified
using His tag systems, and confirmed by SDS-PAGE. Results
of SDS-PAGE of cell lysates before (7.ane 1) and after (lane
2) purification using a nickel-ahelating column are shown
7.n Fic~- 10A. Results of Western blot analysis of rcIP-10
(SO or 200 ng/~.ane) purified with either anti,Hi.S-tag
antibody (lane 1) and anti-caprine IP-10 antibody (lane 2)
are shown in Fig. 10B. Specific band at about l4kDa was
dEtected.
To check the biological aativ~.ties of rcIP-10, in
,tritro chemotaxis assay was carried vut using bovine B-cell
line, KCT-1, transfocted with the capx~.ne CXCR3 cDNA. CXCR3
mRNA expression on the trz~n~s~ected cells was examined by
CA 02497497 2005-03-02
-92-
Noxtharn blot analysis. RNA was extracted from KU-7. cells
that had been transformed with pcDNA3.1.-aa~srine CXCR3
(GXCR3) or parental pcDNA3.l (Mock) (Fig. IOC, left).
Biological activity of rcIP-10 to CXCR3 transEected KU-1
cells expressing (c~.rcle) or not expressing (square) CXCR3
ways tasted by chemotaxXS assay. Bars represent LSM ~ S.E.
The assay demonstrated that CXCR3 txansfectants
consistentJ.y responded to 1 to 20 ng/mI, xcxP-10, but their
responses declined at higher rcYP-10 concentrations,
resulting in a characteristic bell--shaped dose response
curve (Fig. 10C, right). For immunoneutralization
experiments, chemotaxis act~,v~.ty was demonstrated (Fig.
20D) in KU-7. cells with the addition of 20 ng/mh rcrP-10,
to which no further treatment was applied (-), or
neutralized by the pretreatment with the anti~caprine IP-10
antibody (anti IP-10, 30 ~ g/mL), ox with control rabbit ZgG
(contxo~. ZgG). An asterisk indicates a signi.~icant
di.ffexence (p < 0.05). The immunoneutralization
experiments revea~.ed that an anti-TP-~.0 antibody reduced
the chemotactic activity of rcIP-10.
CExpression of zP-10 within goat uterus duxxng early
pregnancy]
Pxeset~ae of ZFN- z in the culture media ( 10 iu, g
protoinx/lane) dorived from Days 14, 17, and 20 goat
conceptus~s was examined by Western blot analysis. The
production of TFN-s protein was the highest on Day 7.7 of
pregnancy (Fig. 7.~.A). Fig. L1B shows results of Northern
blot of endometrial IP-10 mRNA. RNA (20 ~cg/l,ane) from the
conceptus (Day 17: Con) and endometrium (Day 19: D14, Day
17: D17, and Day 20: D20) of pregnant goat (left panel),
and cyclic goats (Day 19, x~.ght panez) that had been
stimulated with rcIFN--z for 24 hours were electrophoresed
on a 1.25 agarose gel. The Northern bJ.ot analysis revealed
that xP-10 mRNA level in the caprine endometrium began to
increase from day ~.7 of pregnancy, and that the conceptus
faGtar xfN-s stimulated IP-~.0 mRNA 1~vel by day 1.4 cyclic
CA 02497497 2005-03-02
- 93 -
endometx~.um in vitro (Fig. 11H). Fresenc~ of IP-10 protein
in the uterine flushing media (10 ,ug protein/lane) ~rom
cyc~.~.c and days 14 and 1'7 ox pregnancy (n = 3 each) were
detected using Wa~t.ern blot analysis k'ig. 11C, left).
Results of densi.tometric analysis of Western blots of IP-~,0
are shown in Fig. 11C, right. Dars represent LSM ~ S.E.,
and an asterzsk indicates a significant difference (p
0.03) when compared with the value from Day cyclic uteri.
It appeared that the uterine flushing media from Day 17
pregnant goats possessed more zP-10 than those of other
days examined. Results of in situ hybridization analysis
of ZP-10 mRNA in the caprxne uterus are shown in Fig. 11D.
Panels a, b, and c represent DIG-labeled antisense capxine
IP--10 cRNR on Day 14 cyclic, Day 14 pregnant, and Day 17
pregnant goats, respectively. Panel d is sense-zP-10 on
Day ~.7 pregnant goats. Here, le: Luminal epithelium, st:
subepithelial stxoma, tr; trophoblast, and scale bar: 5Q 1~m-
TP-10 mRNA was observed in the subepithel~.aZ stroma regions
of pregnant endometrium, and the intensity was greater in
Day 17 pregnant endometr~.um than that of in Day I4 cyclic
or pregnant endometr~.um (fig. 11D).
[Expression and cellular localization of GXGR3 in the
caprine aanaeptus]
Express~.o1-i of GXGR3 mRNA in Days 14, 17, and 20
capr~.ne endametia (D14, D17, and D20, n = 3) and Day 17
conceptus tissues (Con, n = 3) was examined using RT-PGR
(Fig. 12A). Furthermore, the expression of CXCR3 mRNA in
Days 17 and 20 conceptuses (2 ~ g poly(A)'~ RNA/lane, n = 3
far each day) was confirmed by Northern blot. ana7.ys~,s (Fig.
12B) . Immunofluorescence al~alys~.s Was performed using
frozen conceptuses and anti-CXCR3 antibody. Results using
antihuman CXC~t3 monoclonal antibody (a), normal mouse IgG
(n~gative control, d), and nuclei sta~.ned w~,th propi.dium
iodide ware shown in the same field (b and e), and combined
fluorescence image resulting from anti.-CXGR3 and propidium
iodide (c) aro shown in Fig. 12C. Scale bar represents 100
CA 02497497 2005-03-02
um. lA,patchy fluorescence associated with the txophoblast
layer was found and the localization was clearly different
from nuclei.
[Binding of rcrP-~.0 to the caprine trophoblast celXs]
Recombinant proteins, rcZF-10, GST, and caprine
lymphotaatin (hymphotactin-GST) were biotinylated and the
labeled proteins were detected with straptavidin-
harseradish peroxidasea (Fig. 13A). Expressions of IP-10
receptor, CXCR3, mRNA and lymphotacti.n receptor, XCRl, mRNA
in the Day 27 caprine conceptus (Con), endometx~.um (Endo),
ox peripheral blood mononuclear cells (PBMCs) we7:o
confirmed by RT-PCR (Fig. 13g). T~ymphotactin belongs to
the C chemokine family, and its expression, similar to IP--
1.0, was observed in the goat endometrium during early
pregnancy. However, unlike CXCR3, the expression o~
lymphotactin xecegtor, XCR1, mRNA was not detected in the
conceptus. For these reasons, lymphotaatin/XCR was used as
a negative control. Biotinylated proteins wexe incubated
with Day 17 caprine conceptuses and visualized with
steptavidin-horseradish peroxidases. The presence of CXCR3
was observed in the trophoblast cells incubated with only
rclP-10 (Fig. 13C). Scale bar represents 200 ~.cm.
(Stimulation of rclP-10 on the migxat~.on of CXCR3-
expressing conceptus cells]
fig. ISt.A shows results of Northern blot ana7.ysxs
of ~,NAs extracted from HTS-1 cells (-), HTS-1 transf~cted
with empty plasmid (Mock), and HTS,1 transfected with
caprine CXCR3 cDNA (CXCR3). HTS-1 cells were subjected to
che~notaxxs assay. HTS-1 (square) transfected wzth the
empty pxasmid did not respond to rcIP-10. Contxaxxl.y, HTS-
1 transfected with CXCR3-express~.ng pXasmid consistently
responded to ~, to ?o ng/mL rclP-~Ø However, the responses
declined as rcIP--10 concentrations increased, resulting in
a chaxaGteristic bell-shaped dose response cuxve (Fig. 14A,
right). Effect of xczP-1Q on migratory activity of HTS-1
CA 02497497 2005-03-02
-9S-
cells that had been transfected (b~.ack bar) or not
transfected (wha.te bar) with the CXCR3-express~.ng plasmid
was examined (Fig. 14B). These HTS-1 cells were treated
with rclp--~.0 (20 ng/mL), which had been pxetrez~ted with 30
~ g/mL of the anti-c=P-10 a~stibody (Abs : IP--10 ) or 30 ~ g/mD
of normal rabbit IgG (Abs: zgG). Ratio of migrated cells
was calculated as the number of migrated cells treated with
rclP-10 divided by the number of migrated cells without the
rclP-ZO treatment. An aaterir~k indicates a significant
difference (p c 0.05). An anti-rcIP-10 antibody reduced
the chemotactic activity of xcZP-10. Next, primary
trophob~.ast cells expressing CXCR3 on bay 17 of pregnancy
were subjected to the chemotaxis assay. The migration of
trophoblast ce7.~.s was stimulated by rcIP-10 protein, z~nd
the chemotactic activ~.ty wr~s neutralized with the use of an
anti-IP-10 antibody (Fig. 14C).
[Effect of rcZP-10 on the adhesion of trophoblnxt cells to
fibroneetin and endometria~ epithelial cells]
Resu7.ts of the adhesion assay of capririe
trophoblast cells (Day 17 of pregnancy) stimulated (+) or
not stimuJ.ated (-) with rcIP--10 (20 ng/mL) to the plate
coated with collage T, fibroneetin, ox non-subs'Crate showed
that the binding rate of rclP-10-stimulated cells to the
fibronectin-coated plates waa aonsidarably higher than that
of others (fig. 15A). Blocking experiment was pexfoxme~d by
adhesion assay of trophoblast cells to fibronectin using
the pretreatment of rclP-10 with anti--cIP-10 ant~.body (ZP-
10) or noxma~, rabbit xgG (TgG) (Fig. 15B). Fifty mM of
Arg-Gly-,i~sp (RGD) peptide (which inhibits cell adhesion
occurring through RGD sites of fibronectin), 5 mM of Arg-
Gly-Glu (RGE) p~ptide (inactive control.), ox 5 mM of EDTA
(inhibitor of i.ntegrin signaling), were applied to the
trophoblast cells, which were then subjected to chemotaxis
assay (Fig. 158). The RGD peptide decreased the adhesion
of rcIP-10-stimulated cel7.s to fibronectin, but did not the
RGE peptide. EDTA also decreased the adhes~.or~ of
CA 02497497 2005-03-02
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trophoblast cells to fibronectin.
In HTS,1. cells (black bar) transfected with CXCR3
cDNi~, the adhesion of cells of fibronectin was stimuXated
by rcTP--~.D protein and inhibited by anti.~zP-1D anta.body
(Fig. 15C). Tha addition of RGD peptide ox EDTA inhibited
tha adhesion of cells transfected with CXCR3 to fibronectin
(fig. 15C). The RCD peptide and EDTA treatments inhibited
migration activ~.txes of cells (white bar) transfected with
empty plasmids.
HTS-1 cells (black bar) transfected with CXCR3
plasmid or HT5-1 cells (white bar) transfected with empty
plasmid were subjected to the adhesion assay with
endometria~, epithelial cells (Fig. 15D). The adhesion of
CXCR3-expressing HTS-1 to the endametrial epithelial cells
was increased by tclP-~.0 protein and inhibited by the use
of antibody to the TP-10 in the neutxal.~.z.ing experiment.
EDTA-treated trophob7.ast Cells lost the adhesion activity
to epithel~.a~, cells. RGD peptide inhibited the adhesion
activity. Bars represent T,SM t S.E., and an asterisk
indicates a significant difference (p < 0.05).
[Expression of integrin subunits in the capxine trophoblast
cells stimulated by xcZP~10]
The Expression of integrin subunits was examined
in HTS-1 cells stimulated by rare-1D. Transcripts for
integrin subunits, a 5, ~ V, ~ ~., (3 3, and l3 5 were detected
by us~.ng RT-PCR. The expression of integrin a 5, ~ V, and
(33 subunits mRNA in trophoblast cells were stimulated by
rcIP--10, and the stimulation was d~.m~.n~.shed by the
neutralization with the anti-IP-10 antibody (Fig. 16). The
expression of integrin /31 and j35 subunits mRNA was not
influenced by rcTP-10. Bars represent LSM ~ S.E.
xhe results above show that expressions of TP-20
arid xFN- y mRNA time-varyingly and cell--specifically occur
during eaxly pregnancy in the caprine uterus as well as in
the ovine uterus, and conceptus 2FN-~c has an abxZi.ty to
rtgulate endometrial IP-10 expression. These f~.nd~.ngs
CA 02497497 2005-03-02
_97_
suggest that IP-10 zegulated by IFN-s plays an important
role in the recruitment and/or redistribution of a.mrnune
cells during early pregnancy.
The pxesont invention provides a way for
~.nvestigating relationship betweon IE-10 and IFN--t and IP-
1Q functions in uterus.
~NDUSTR=RL APE,TrZCABIIrITY
Tho prosent invention provides a technology foz
utilizing IP--10, which contxo~.s implantation of conceptus
or i.ts process, and the present invent~.on provides
pharmaceutical drugs, veterinary drugs, therapeutic methods,
activity-measuring methods, assay methods, and xaagonts
used therefore, wh~.ch control or utilize the maternal
systom in the pregnant recognition process. According to
the present invention, pregnancy can be securely promoted
or prevented in animals including human and domestic
animals, and usoful materials or medicine for such puxpdses
can be readxJ.y developed.
Whilct specific details of the present invention
have been dosaribed in terms of preferred embodiments and
examples, it will be apparent to those of skill in the
field of the art that variations may be applied to those
disclosed in the foregoing. In light of the disclosure,
various modifications and rearrangements which can be made
to those set forth.herein are deemed to be w~.thin the
spirit and scope of the appended claims.
<Free text sequence>
SEQ ID NO: 3, Description of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
SEQ TD NO: 4, Descx~.ptXOn of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
5EQ TD NO: 5, Descx~.ptxon of Artificial Sequence:
Oligonucleotide to act as a primer for pCR
SEQ ID NO: 6, Description of Artificial Sequence:
CA 02497497 2005-03-02
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07.xgarsucleotide to act as a primer iox PCR
SEQ ID NO: 7, Description of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
SEQ TD NO: B, Description of Artificial Sequence:
Oligonuclcot~.de to act as a primer for PCR
SEQ ID NO: 9, Description of Artificial Sequence:
OligonucXeotide to act as a primer for PCR
SEQ ID NO: 7.0, Description of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
SEQ ID NO: 7.1, Description of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
SEQ ID NO: 12, Descxipt~.on of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
SEQ ID NO: 13, Description of Artificial Sec~xenae:
Oligonucleotide to act as a primer for PCR
SEQ ZD NO: 14, Description of Artificial Sequence:
Oligonucleotide to act as a px~.mer far PCR
SEQ ID NO: ~.5, Description of Artificial Sequence:
OligonucXeota.de to act as a primor for PCR
SLQ TD NO: 16, Description of Artificial Sequence:
Oligonucleotide to act as a primer for PCR
SEQ ID NO: 17, Description of Axt~.f~.c~.aJ. Sequence:
Oligonucleotide to act as a primer for ECR
SEQ ID NO: 18, Description of Artificial Sequence:
Oligonucleotide to set as a primer far PCR
SEQ ZD NO: 19, Description of Artificial Sequence:
0ligonucleotide to act as a primer for pCR
sEQ ID NO: 20, Description of Artificial sequence:
ol,igonucleotide to act as a primer for PCR
SEQ ID NO: 21, Description of Artificial Sequence:
Oligonucleotide to act as a primer far PCR
SEQ TD NO: 22, Descxipt~.on of Axt~.f~.c~.al Sequence:
oligonuclevtide tv act as a primer for PCR
SEQ ID NO: 23, Description of Artificial Sequence:
Oligonucleotide to act as a primer fox PCR
SEQ TD NO: 24, Description ofi Artificial Sequence:
Oligonucleotide to act as a primer for PCR
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SEQ ID NO: 25, Description of Artificial Sequence:
Oligonuclaotids to act as a primer for PCR
SEQ ID NO: 26, D~~Criptaon of ArtificiaX Sequence:
Ol~.goriticleotide to act as a pxa.mer for PCR
CA 02497497 2005-03-02
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SEQUENCE LISTING
<x10~ ProteinBxpress Co., Ltd.
<120> Regulator for Implantation
<130> PE~OIpCT
<150> JP 2002-259268
<151> 2002-9-4
<160> 26
<170> PatentIn Ver. 2.0
<210> 1
<211> 1171
<2X25 DNA
<213> Ovls arias
<220>
<221> CDS
<222> (60)..(368)
<400> 1
cactcctcaa ctcttcaggc agtctgagct actgcagaag taccttcagt tgcagcacc 59
atg aaC aaa agt ggt ttt ctt att ttC tgC ctt atc ctt ctg act ctg 107
Met Asn Lys Ser GIy Phe Leu IIe Phe Cys Leu Ile Leu Leu Thr Leu
1 5 10 15
agt caa ggc ata cct ctG tct agg aaC aca cgc tgc aCC tgC.aLC gag 155
Ser Gln Gly IIe Pro Leu Ser Arg Asn Thr Arg Cys '1'hr Cys Ile Glu
20 25 30
atc agt aat ga tct ,gtt aat cca agg tcc tta gaa aaa ctt gaa ctg 203
Ile Ser Asn ~ly Ser Val Asn Pro Arg Ser Leu GIu LYS Leu Glu Leu
35 40 45
att cct gca agt caa tcc tgc cca Ggt gtC gag att att gCC aCa atg 251
Ile Pro Ala Ser Gln Ser Cys Pro Arg VaI Glu Ile Ile Ala Thr Met
50 55 60
aaa agg aat ggg gag aaa aga tgt Ctg aat CCa gaa tCt aag acC atc 299
Lys Arg Asn GIy GIu Lys Arg Cys Leu Asn Pro Glu Ser Lys Thr Ile
65 70 75 80
aag aat tta Ctg aaa gCa att aac aag Caa agg act aaa aga tct cct 34?
Lys Asn Leu Leu Lg~ Ala lle Asn Lys G9n0 Arg Thr Lys Arg Sg5 Pro
cga aca cag aaa gag gca taa tcactgcact aCtgataaga tggaCCagag 39$
Arg Thr Gln Lys Glu Ala
100
CA 02497497 2005-03-02
z~s
agaagctacc tctacaattg tttccctgtg tacagtatat gtcaagccct aattgttcgt 458
ggacttcagt tctcctaaaa ggtgaccaag ccagtcacca aatcagctgc tactactcCt 518
gcagggggag ggtggctcat ca.ccctgagc tgttcagtag tgactctgcc ctggcactgt 578
gactgtaagc tataccgggg cgctacgttc tca.gttaatg tgctaagtcc cagccttgct 638
actgacagct tcttcccctt tccaatcttt ctaggttatt aagggatctt tccagctctg 698
ggcttattag agaccttagg atctcaaata actaagagac attcaaacca ataatgcaat 758
ctgcttttta aagaaagatc tttactccag gggcttcact gccatccCtc caaggggccc 818
gtatCCtttc aggtgttatg tacatagttc caaatataca gaagcagcca gaaatatctg 878
gaaatgtagg tctaaacagt attacttagt caaaaactat acaaagtaga attcttgaag 93$
atatatgttt cttatatgat tttcagtgtt catggaataa cttgtataca actatCaaCt 998
tatgtaatta ttgcaatgga ataaattttt aaatttagat acatgttctg caggctatgt 1058
aagacaaata tgctaaatgc tttccaaaat aaaagtaatg ttctctccca gaaatactaa 1118
gaaagattat gtaattgttt tagaggccaa aaacataata aataattata act 1171
<210> 2
<211> 102
<212> PRT
<213> 0vis aries
<400> 2
Met Asn Lys Ser Gly Phe Leu Iie Phe Cys Leu Ile Leu Leu Thr Leu
1 5 10 15
Ser Gln Gly Ile Pro Leu Ser Arg Asn Thr Arg CYS Thr Cys Ile Glu
20 25 30
Ile Ser Asn Gly Ser Val Asn Pro Arg Ser Leu Glu Lys Leu Glu Leu
35 40 45
Ile Pro Ala Ser Gln Ser Cys Pro Arg Val Glu lle lle Ala Thr Mct
50 55 GO
Lys Arg Asn Gly Glu Lys Arg Cys Leu Asn Pro Glu Ser Lys Thr Ile
65 70 75 80
Lys Asn Lea Leu Lys Ala Ile Asn Lys Gln Arg Thr LYS Arg Ser Pro
85 94 95
Arg Thr Gln Lys Glu Ala
100
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<21.0> 3
~211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Seguence:0ligonucleotide
to act as a primer for FCR
<400> 3
cactcctcaa ctcttcaggc 20
<210> ~:
<211> 20
<212> DNA
t213> Artificial Seauence
<220>
<223> Description of Artificial Se4uence:Oligonucleotide
to act as a primer for PCR
<400> 4
CCSttCCttt tCattgtgBC 2a
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:Oligonucleotide
to act as a primer for PCR
«:00~ 5
gcaCcagctt cgatcggtac 20
t210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial SEquence:Oligonucleotide
to act as a primer for PCR
<400> 8
gatgcgggcg tagcaatagg 20
CA 02497497 2005-03-02
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<210> ?
X21. X > 20
<212> DNA
<2I3> ArtIfIcial Sequence
<2207
<223> Description of Artificial Sequence:Ollgonucleotide
to act as a primer for PCR
<400> 7
catcttcccc atggccttcg 20
<2I0> 8
<211> 20
<212> DNA
<2I3> Artificial Sequence
<220>
<223> Description of ArtlfIclal Sequence:Ollgonucleotlde
to act as a primer for PCR
<400> 8
tcatctcaaa gtgagttca8 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
G220>
<223> DcscrIptlon of ArtIfIcIal Soquence:Oligonucleotide
to act as a primer for PCR
«;00> fl
Cgatgaaata cacaagctcc 20
<210> 10
<211> 20
<212> DNA
<213> ArtIfIcial Sequence
<220>
<223> Description of Artificial Sequence:0ligonucleotide
to act as a primer for PCR
<400> 10
gattacattg at~ctctccg 20
<210> 11
<2J.X> 20
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<212> DNA
<213> Artificial Sequence
<220>
<228> Description o~ Artificial Sequence:Oligonucleotide
to act as a primer for PCR
<400> 11
atggggaagg tgaaggtcgg 20
<210> 12
<21I> 20
<212> DNA
<213~ Artificial Sequence
<220>
<223> Description of Artificial Sequence:Oligonucleotlde
to act as a primer for PCR
<400> I2
atgtgggcca tgaggtccac 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<22Q>
<223> Description of Artificial Sequence:Oligonucleotide
to act as a primer for PCR
«,00> X3
atggggaagg tgaaggtcgg 20
<2I0> I4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:0ligonucleotide
to act as a primer for PCR
<400> 14
atgtgggcca tgaggtcca.c 20
<2I0> I5
<21I> 20
<212> DNA
<21.3> Artificial Sequence
CA 02497497 2005-03-02
~ ~ 8
<220>
<223> Description of Artificial Sequence:OlIgonucleotide
to act as a primer for PCR
«00 > 15
atggagccct cagacatccc 20
<210> x6
<211> 20
<212> DNA
C213> Artificial Sequence
C220>
<223> Description of Artificial Sequence:Oliganucleotide
to act as a primer for PCR
C400> x6
gaggatctcc acgtagcaga 20
<2105 17
C2II> 20
<2I2> DNA
<213> Artificial Sequence
<Z20>
C223> Description of Artificial Sequence:0ligonucleotide
to act as a primer for PCR
<400> 17
tgctgtgaac cagagtcgtc 20
<2Ia> Is
C2II> 2a
<212> DNA
<2X3> Artificial Se4uence
<220>
C223> Description of Artificial Seauence:Oligonucleotide
to act as a primer for PCR
<400> 18
atccactgca cagctgtggc 20
<210> I9
C21I> 20
C212> DNA
<213> Artificial Sequence
<220>
CA 02497497 2005-03-02
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<223> Description of Artificial Sequence:Ollgonucleotlde
to act as a primer for PGR
<400> 19
gaagcaggaa agagagCCtg 20
<210> 20
<21I> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:Oligonucleotide
to act as a primer for PCR
«00> 20
ctatatcc~t ggctcctttc 20
<210> 21
<2I1> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:0ligonucleotide
to act as a primer for PCR
<400> 21
ctcaaatcca gccaCagcag 20
<210> 22
<2117 20
<212> UNA
<213> Artificial Sequence
<220>
<2237 Description of Artificial Sequence:Oligonucleotide
to act as a primer for PCR
<400> 22
ccagcgaa~t gaaacacagc 20
<210> 23
<21,1> 20
<212> DNA
<2I3> Artificial Sequence
<220>
<223> DeserIptlon of Artificial Sequence:Oligonucleotide
to act as a primer far PCR
CA 02497497 2005-03-02
$ ~ $
<400> 23
agattggaga cacggtgagc 20
<210> 24
<211> ZO
<212> DNA
~2X3> Artificial Sequence
<220>
<223> Description of Artificial Sequence:Oligonucleotide
to act as a primer for PCR
<4007 24
gtacttgaaa gtgatcttgc 20
<210> 25
<211> 20
<2X2> DNA
<213> Artificial Sequence
<220>
<228> Description of Artificial Sequence:Oligonucleotide
to act as a primer for PCR
<~100> 25
gtctgaagat tggggacagc 20
<210> 26
<2X1~ 20
<212> DNA
<213> Artificial Sequence
<220>
<223> DeSGription of Artificial Sequence:0lIgonucleotIde
to act as a primer for PCR
<400> 28
ggtaCacgct Ctggttctcc 20
